Methods and compositions for the treatment of peripheral artery disease

ABSTRACT

Compositions and methods for treating peripheral artery disease in a patient are provided. Compositions comprise recombinant fibroblast growth factor-2. Fibroblast growth factor, such as FGF-2, is administered in therapeutically effective amounts to treat or prevent peripheral artery disease including claudication and critical limb ischemia. Pharmaceutical compositions comprising a therapeutically effective amount of FGF-2 and a pharmaceutically acceptable carrier are also provided. The methods of the invention to treat peripheral artery disease and claudication comprise administering at least a single dose of a pharmaceutical composition comprising the FGF, such as FGF-2, via intra-arterial, intravenous, or intramuscular infusion to the patient. It is recognized that increased benefits may result from multiple dosing, including intermittent dosing.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication Serial Nos. 60/213,504, filed Jun. 22, 2000, 60/264,572,filed Jan. 26, 2001, and 60/276,549, filed Mar. 16, 2001, each of whichis entitled “Methods and Compositions for the Treatment of PeripheralArtery Disease,” the contents of which are herein incorporated byreference in their entirety.

FIELD OF THE INVENTION

[0002] The invention relates to methods and pharmaceutical compositionsfor treating peripheral artery disease, particularly the administrationof compositions that contain recombinant fibroblast growth factor-2(rFGF-2).

BACKGROUND OF THE INVENTION

[0003] Coronary artery disease (CAD) and peripheral artery disease (PAD)are conditions characterized by insufficient blood flow, usuallysecondary to atherosclerosis. Symptoms of ischemia (angina pectoris forCAD or intermittent claudication for PAD) are brought on by stress andrelieved by rest. In CAD, symptoms may become life threatening due tomyocardial infarction, arrhythmia, and progressive heart failure. InPAD, symptoms are less likely to be life threatening except whencritical limb ischemia develops, but the risk of adverse cardiovascularevents and death is increased.

[0004] Identification and management of risk factors are important inthe medical management of both CAD and PAD. Pharmacologic management ofrisk factors may include anti-hypertensives, lipid-lowering agents, andhypoglycemic agents; smoking cessation, diet, and exercise are oftenprescribed with variable compliance. Pharmacologic management aimed atreduction of symptoms of ischemia often includes vasodilators,anti-anginal, and anti-platelet therapy. Mechanical revascularization bypercutaneous angioplasty (with or without a stent) and direct surgicalreconstruction improve blood flow and reduce symptoms. However,restenosis after angioplasty and progression of disease may limit theduration of the benefit.

[0005] PAD afflicts approximately 11 million patients in the UnitedStates. Approximately one third of these patients experienceintermittent claudication (discomfort, pain, fatigue, or heaviness inthe leg muscles that consistently is brought on by the same amount ofmuscular activity and relieved by rest). Claudication is similar toangina and represents ischemic muscle pain that may be localized to thehip, buttock, thigh, or calf. It occurs predictably with the same amountof physical stress. Atherosclerosis is systemic, but often one lowerlimb is more affected than the other. Patients may develop critical limbischemia, with rest pain, non-healing ulcers, and/or gangrene. Rest painoccurs when blood supply is inadequate to meet the basic nutritionalrequirements at rest and typically localizes in the toes or foot of theaffected limb.

[0006] The prevalence of CAD and PAD is expected to increase incountries with aging populations, as aging is a primary risk factor foratherosclerosis. Less invasive catheter-based treatment methods and morecost-effective programs and treatment methodologies are needed to managethese conditions.

SUMMARY OF THE INVENTION

[0007] Compositions and methods for treating peripheral artery disease(PAD) in a patient are provided. Pharmaceutical compositions comprisinga therapeutically effective amount of fibroblast growth factor, such asFGF-2, and a pharmaceutically acceptable carrier are provided. Suchcompositions when administered in accordance with the methods of theinvention provide effective treatment for PAD patients including thosesuffering intermittent claudication associated with this disease. Suchcompositions may also be administered to PAD patients to preventprogression of critical limb ischemia to amputation.

[0008] The methods of the invention comprise administeringpharmaceutical compositions comprising a therapeutically effectiveamount of a growth factor, such as FGF-2, as an intra-arterial infusion(IA), intravenous infusion (IV), intramuscular injection (IM), orsubcutaneous injection (SC). A single-dose administration of FGF-2 isefficacious for the treatment of PAD. Therapeutic benefits may beobtained with multiple doses without compromising safety. Administrationof FGF-2 improves peak walking time in patients with PAD for at least 90days after FGF-2 administration. FGF-2 can be used to treat patientssuffering from critical limb ischemia including those with resting painwith and without non-healing ulcers. Additionally, FGF-2 can be used totreat PAD patients suffering from critical limb ischemia. TheFGF-containing composition of the invention can be administered asadjuncts to vascular surgery involving mechanical bypass andpercutaneous transluminal interventions with balloon catheters, with orwithout stents.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 sets forth the DNA sequence (SEQ ID NO:1) encodingfibroblast growth factor-2 (FGF-2) having the amino acid sequence setforth in FIG. 2; this FGF-2 is of bovine origin. The translated aminoacid sequence (SEQ ID NO:2) is also shown.

[0010]FIG. 2 sets forth the amino acid sequence (SEQ ID NO: 2) for the146 amino acid residue bovine FGF-2 encoded by the DNA sequence setforth in FIG. 1.

[0011]FIG. 3 sets forth the DNA sequence (SEQ ID NO:3) encoding thetranslated amino acid sequence (SEQ ID NO:4) for the 146 amino acidresidue FGF-2 of human origin.

[0012]FIG. 4 sets forth the DNA sequence (SEQ ID NO:5) encoding thetranslated amino acid sequence (SEQ ID NO:6) for the 155 amino acidresidue FGF-2 of bovine origin.

[0013]FIG. 5 sets forth the DNA sequence (SEQ ID NO:7) encoding thetranslated amino acid sequence (SEQ ID NO:8) for the 155 amino acidresidue FGF-2 of human origin.

[0014]FIG. 6 shows the relative change in peak walking time (PWT) at day90 with administration of rFGF-2 in patients in a phase II clinicalstudy. In this study, three patient groups were assessed: a groupadministered a placebo on both days 1 and 30; a group administered asingle dose of rFGF-2 (30 μg/kg) on day 1 and a placebo on day 30; and agroup administered a dose of rFGF-2 (30 μg/kg) on both days 1 and 30.The mean and standard error are indicated for the measured PWT in eachof these groups. The ANOVA analysis excluded patients with missing dataand revascularized patients. The ANOVA of Ranks test included patientswith missing data and revascularized patients by assigning the lowestrank. Pairwise comparison indicated a p value of 0.026 between thesingle dose and placebo groups and a p value of 0.45 between the doubledose and placebo group. The figure provides the primary efficacyanalysis of the clinical trial, which specified the use oflog-transformed data. This is considered appropriate statisticalmanagement of data when the results have skewness or kertosis such as isoften seen in treadmill tests.

[0015]FIG. 7 shows absolute change in PWT at days 90 and 180 for thepatient groups receiving placebo, single-dose rFGF-2, or double-doserFGF-2. For each patient the PWT at baseline is subtracted from the PWTat day 90 and the differences are summed for each group and a meandetermined; the data are analyzed by an analysis of variance (ANOVA).

[0016]FIG. 8 shows the percent absolute change in PWT in the threepatient groups shown at day 90 and day 180. The percent change in PWTaveraged across the two rFGF-2 groups is also shown (designated AnyFGF).

[0017]FIG. 9 shows the measured ABI (ankle brachial index) for the threepatient groups of the phase II clinical study. A baseline measurement, aday-90 measurement, and the corresponding change between the baselineand day-90 measurement are indicated. The mean change in ABI is alsoshown for the three patient groups. The ABI is described in An OfficeBased Approach to the Diagnosis and Treatment of Peripheral ArterialDisease (2000) Society of Vascular Medicine and Biology (MedicalCommunications Media, Inc., Wrightstown, Pa.) herein incorporated byreference. Subjects having an ABI>1.2 at baseline are excluded from theanalysis.

[0018]FIG. 10 shows the results of the WIQ severity of claudication forthe three patient groups in the phase II clinical study at day 90 andday 180. Values represent the percentage of patients in each groupindicating an improvement, no change, or worsening of this condition.

[0019]FIG. 11 shows the severity scores at baseline, day 90, and day 180for distance, speed, and stair climbing for each group. The figuredemonstrates that the results for the single-dose group were better thanthe results for the placebo group for WIQ distance, speed, and stairclimbing. The figure is shown with a scale where higher scores arebetter.

[0020]FIG. 12 depicts the physical summary scores from the short form 36(SF-36). A change of 1 point is associated with an increased lifespan of2 years. The change scores in the figure indicate an improvement in thesingle-dose group versus the placebo group by greater than 2 points atday 90.

[0021]FIG. 13 summarizes the results of the study.

[0022]FIG. 14 shows the measured ABI (ankle-brachial index) for thethree patient groups of the phase II clinical study, when subjectshaving an ABI>1.2 at anytime (i.e., baseline, day 90, and/or day 180)are excluded from the analysis. A baseline measurement, a day-90measurement, and the corresponding change between the baseline andday-90 measurement are indicated. The mean change in ABI is also shownfor the three patient groups.

[0023]FIG. 15 shows a hypothetical plot of peak walking time at day 90(PWT90) versus peak walking time at baseline (PWTB) when absolute changescore is assumed to be the correct analysis variable. Assumptions:(PWT90−PWTB)=1d, then PWT90=1.0*PWTB+d PWT90, scatter plot is linear,slope=1.0, and intercept is d (unrestricted).

[0024]FIG. 16 shows a hypothetical plot of PWT90 versus PWTB whenrelative change score is assumed to be the correct analysis variable.Assumptions: (PWT90/PWTB)=1d, then PWT90=1d*PWTB+0.0 (across the fullrange of PWTB), scatter plot is linear, slope is Id (unrestricted), andintercept is 0.0.

[0025]FIG. 17 shows a scatter plot of PWT90 versus PWTB plus anunrestricted spline regression curve for the placebo (Δ), single-dose(□), and double-dose (∘) groups.

[0026]FIG. 18 shows the same scatter plot from FIG. 16 plus curvesrepresenting regression model 2 described in Table 15 as applied to theplacebo (Δ), single-dose (□), and double-dose (∘) groups. P=placebo;S=single-dose; D=double-dose.

[0027]FIG. 19 shows the scatter plot of PWT180 versus PWTB plus anunrestricted spline regression curve for the placebo (Δ), single-dose(□), and double-dose (∘) groups.

[0028]FIG. 20 shows the effect of single administration byintra-arterial infusion (IA) or intramuscular injection (IM) and 14-daycontinuous intra-arterial infusion on total hindlimb blood flow in a ratbilateral PAD model. Phosphate-buffered solution (PBS) served as thevehicle control.

DETAILED DESCRIPTION OF THE INVENTION

[0029] One potential new alternative for the treatment of intermittentclaudication due to peripheral artery disease (PAD) is the use ofangiogenic growth factors that promote the formation of new bloodvessels from preexisting ones (angiogenesis) and also restoreendothelial cell function. In angiogenesis, endothelial cells leavetheir resting state and start to digest the underlying basement membranefollowed by proliferation, migration, and finally formation of a hollowtube (Gerwins et al. (2000) Crit. Rev. Oncol. Hematol. 34(3):185-194).Fibroblast growth factors bind to cell surface receptors that areligand-stimulatable tyrosine kinases. Binding of these growth factors totheir receptors leads to activation of the intrinsic tyrosine kinase andsignal transduction to downstream signaling cascades (Gerwins et al.(2000) Crit. Rev. Oncol. Hematol. 34(3):185-194). Angiogenesis inischemic tissues can be promoted by the transmural delivery ofangiogenic growth factors such as VEGF, FGF, and PDGF using anintravascular infusion catheter. See, for example, U.S. Pat. No.5,941,868.

[0030] Compositions and methods for treating PAD in a patient areprovided. The compositions and methods are useful in the treatment andprevention of claudication and critical limb ischemia due to PAD. Theterm “critical limb isehemia” is used for all patients with chronicischemic rest pain, ulcers, or gangrene attributable to objectivelyproven arterial occlusive disease. The term “critical limb ischemia”implies chronicity and is to be distinguished from acute limb ischemia.By “acute limb ischemia” is intended any sudden decrease or worsening inlimb perfusion causing a threat to extremity viability. See, J. Vasc.Surg. 31:S135, S168, herein incorporated by reference. The methods ofthe invention utilize angiogenic agents, such as angiogenic members, ofthe fibroblast growth factor (FGF) family, including preferably FGF-1,FGF-2, FGF-4, FGF-5, FGF-18, and most preferably FGF-2. It is recognizedthat all angiogenic growth factors herein described may be recombinantmolecules. Also, it is recognized that compositions of the invention maycomprise one or more fibroblast growth factors as angiogenic agents aswell as biologically active variants thereof. Variants of an FGFsequence include, but are not limited to, angiogenically activefragments, analogues, and derivatives. By “fragment” is intended apolypeptide consisting of only a part of the intact FGF sequence andstructure, and can be a C-terminal deletion, N-terminal deletion, orboth. By “analogues” is intended analogues of either the angiogenicagent FGF or fragment thereof that comprise a native FGF sequence andstructure having one or more amino acid substitutions, insertions, ordeletions. Peptides having one or more peptoids (peptide mimics) andmuteins, or mutated forms of the angiogenic agent, are also encompassedby the term analogue. By “derivatives” is intended any suitablemodification of the angiogenic agent, fragments of the angiogenic agent,or their respective analogues, such as glycosylation, phosphorylation,or other addition of foreign moieties, so long as the angiogenicactivity is retained. Methods for making fragments, analogues, andderivatives are available in the art. See generally U.S. Pat. Nos.4,738,921, 5,158,875, and 5,077,276; International Publication Nos. WO85/0083 1, WO 92/04363, WO 87/01038, and WO 89/05822; and EuropeanPatent Nos. EP 135094, EP 123228, and EP 128733; herein incorporated byreference.

[0031] Such variants should retain angiogenic activities and thus be“angiogenically active.” The variants may be measured for angiogenicactivity using standard bioassays. Representative assays include knownradioreceptor assays using placental membranes (see, e.g., U.S. Pat. No.5,324,639; Hall et al. (1974) J. Clin. Endocrinol. and Metab.39:973-976; and Marshall et al. (1974) J. Clin. Endocrinol. and Metab.39:283-292). Additional assays include mitogenic activity as determinedin an in vitro assay of endothelial cell proliferation. This activity ispreferably determined in a human umbilical vein endothelial (HUVE)cell-based assay, as described, for example, in any of the followingpublications: Gospodarowicz et al. (1989) Proc. Natl. Acad. Sci. USA87:7311-7315; Ferrara and Henzel (1989) Biochem. Biophys. Res. Commun.161:851-858; Conn et al. (1990) Proc. Natl. Acad. Sci. USA 87:1323-1327;Soker et al. (1998) Cell 92:735-745; Waltenberger et al. (1994) J. Biol.Chem. 269:26988-26995; Siemmeister et al. (1996) Biochem. Biophys. Res.Commun. 222:249-255; Fiebich et al. (1993) Eur. J. Biochem. 211:19-26;Cohen et al. (1993) Growth Factors 7:131-138. A further biologicalactivity is involvement in angiogenesis and/or vascular remodeling,which can be tested, for example, in the corneal pocket angiogenesisassay as described in Connolly et al. (1989) J. Clin. Invest.84:1470-1478 and Lobb et al. (1985) Biochemistry 24:4969-4973; theendothelial cell tube formation assay, as described for example inPepper et al. (1992) Biochem. Biophys. Res. Commun. 189:824-831; Goto etal. (1993) Lab. Invest. 69:508-517; or Koolwijk et al. (1996) Cell Biol.132:1177-1188; the chick chorioallantoic membrane (CAM) angiogenesisassay as described for example in Pluet et al. (1989) EMBO. J.8:3801-3806; the endothelial cell mitogenesis assay as described inBohlen et al. (1984) Proc. Natl. Acad. Sci. USA 81:5364-5368; Presta etal. (1986) Mol. Gen. Biol. 6:4060-4066; Klagsbrun and Shing (1985) Proc.Natl. Acad. Sci. USA 82:805-809; Gosodarowicz et al. (1985) J. Cell.Physiol. 122:323-332; or the endothelial cell migration assay asdescribed in Moscatelli et al. (1986) Proc. Natl. Acad. Sci. USA83:2091-2095; and Presta et al. (1986) Mol. and Cell. Biol. 6:4060-4066;all of which are herein incorporated by reference. It is recognized thatone or more of the assays may be used. Preferably, the variant has atleast the same activity as the native molecule.

[0032] Fibroblast growth factor-2 (FGF-2), including recombinantlyproduced forms (rFGF-2), is a potent mitogen and angiogenic agent thathas utility for treatment of coronary artery disease (angina) andperipheral artery disease (claudication). Although FGF-2 is normallymade in many body tissues and is involved in the body's response tocertain ischemic conditions, the body's own supply of FGF-2 may not besufficient to circumvent the complications of atherosclerosis andarterial insufficiency/ischemia.

[0033] Compositions and methods of the invention can be used to treatPAD patients, even those suffering a wide spectrum of related clinicalailments, including but not limited to coronary artery disease (CAD),myocardial infarctions, stroke, diabetes, dyslipidemias, hypertension,and patients who have had surgical or catheter-based revascularizations.Fibroblast growth factors, particularly FGF-2, can be used to treat PADpatients suffering from claudication, including those having criticallimb ischemia. Critical limb ischemia, when left untreated, can progressto acute limb ischemia and ultimately necessitate amputation of thelimb. As such, the methods of the invention can be used to prevent acutelimb ischemia.

[0034] The FGF-containing compositions of the invention are administeredintra-arterially (IA), intravenously (IV), intramuscularly (IM),subcutaneously (SC), transmurally, and the like to a patient in needthereof. By “transmural” administration is intended localized deliveryof the composition into the blood vessel or body lumen wall includingneointimal, intimal, medial, advential, and periviascular spaces,particularly adjacent to the target site. By “target site” is intendedthe area surrounding or immediately surrounding the blood supply intothe extremities, e.g, legs.

[0035] Intra-arterial administration (IA) involves delivery of theFGF-containing composition into at least one artery. In an IA infusion,the infusion is typically divided into several arteries in the legs,e.g., the left and right common femoral arteries, but is sometimesadministered into a single artery. The infusion can be administered forabout 1 minute, 1 to 5 minutes, 10 to 20 minutes, or 20 to 30 minutesinto each artery in both legs. The infusion can be repeated from time totime to achieve or sustain the predicted benefit. The timing for repeatadministration is based on the patient's response as measured bysymptoms and hemodynamic measures. A therapeutically effective dose oramount of FGF, such as FGF-2, that is to be given as an infusion can bedivided into two doses, and a single dose administered into each leg ofa patient undergoing treatment. In this manner, the total dose isdelivered such that the angiogenic agent is presented to both legs ofthe patient.

[0036] Thus in one embodiment, a therapeutically effective dose oramount of FGF as defined elsewhere herein is administered via IAinfusion using a bilateral delivery method such that the procedure canbe completed with a single puncture. In this manner, one-half of thetherapeutically effective amount or total dose of FGF, such as rFGF-2,is infused into the common femoral artery of the first leg, followed byguiding the catheter over the bifurcation of the aorta to thecontralateral iliac artery and common femoral artery and then infusingthe remainder of the total dose into the femoral artery of the secondleg. The rate of each infusion, one into each leg, is about 1 mL/perminute over about a 10-minute period, with a short interruption betweenthe first and second infusion. Thus, the second infusion generallybegins within about 1 hour of the first infusion, but can begin up to 2,3, or 4 hours after the first infusion. Preferably the second infusionbegins within about 30 minutes, more preferably within about 20 minutes,even more preferably within about 10 minutes, still more preferablywithin about 5 minutes of the completion of the first infusion. Eachinfusion can take less than about 10 minutes, such as 3, 4, or 5minutes, so long as the FGF is not administered as a bolus. It isrecognized by one of skill in the art that the therapeutically effectivedose or amount of FGF, such as rFGF-2, can be divided between the twolegs of the patient such that unequal portions of the total dose aredelivered to each leg, for example, one-third to one leg, and two-thirdsto the other leg. The advantage of the bilateral delivery method is thatthe two infusions, one into each leg, can be accomplished with a singlepuncture to the subject. In this embodiment, the sight of the punctureis preferably at groin level. A brachial approach may be used if deemedpreferable by the treating physician. With this procedure, the cathetercan be guided more distally, such as in the area just above the knee, aslong as the obstruction to blood flow remains distal to the point ofinfusion.

[0037] Alternatively, the therapeutically effective amount of FGF, suchas rFGF-2, can be delivered by direct IA puncture into each commonfemoral artery. In this manner, one-half of the dose of FGF isadministered into one common femoral artery, while the other half of thedose of FGF is administered into the other common femoral artery. DirectIA puncture can be advantageous in that it avoids the catheterizationprocedure required with bilateral delivery, but it necessitates twopunctures when the therapeutically effective dose is to be divided andinfused into both legs. As with bilateral delivery, each infusion isdelivered at a rate of about 1 mL per minute over about a 10-minuteperiod, with a short interruption between the first and second infusion.Thus, the second infusion generally begins within about 1 hour of thefirst infusion, but can begin up to 2, 3, or 4 hours after the firstinfusion. Preferably the second infusion begins within about 30 minutes,more preferably within about 20 minutes, even more preferably withinabout 10 minutes, still more preferably within about 5 minutes of thecompletion of the first infusion. Each infusion can take less than about10 minutes, such as 3, 4, or 5 minutes, so long as the FGF is notadministered as a bolus. Again, it is recognized that thetherapeutically effective dose or amount of FGF, such as rFGF-2, can bedivided between the two legs of the patient such that unequal portionsof the total dose are delivered to each leg.

[0038] Delivery of the FGF-containing compositions in accordance withthe methods of the invention may be accomplished through a variety ofknown intravascular drug delivery systems. Such delivery systems includeintravascular catheter delivery systems. A variety of catheter systemsuseful for the direct transmural infusion of angiogenic growth factorsinto the blood vessel are well known in the art. For purposes ofpracticing the invention, any of a variety of diagnostic or therapeutictype catheters could be used. Where the FGF is administered inconjunction with an angioplasty, balloon catheters can be used. Ballooncatheters having expandable distal ends capable of engaging the innerwall of a blood vessel and infusing an angiogenic growth factor directlytherein are well described in the patent literature. See, for example,U.S. Pat. Nos. 5,318,531; 5,304,121; 5,295,962; 5,286,254; 5,254,089;5,213,576; 5,197,946; 5,087,244; 5,049,132; 5,021,044; 4,994,033; and4,824,436. Catheters having spaced-apart or helical balloons forexpansion within the lumen of a blood vessel and delivery of atherapeutic agent to the resulting isolated treatment site are describedin U.S. Pat. Nos. 5,279,546; 5,226,888; 5,181,911; 4,824,436; and4,636,195. Non-balloon drug delivery catheters are described in U.S.Pat. Nos. 5,180,366; 5,112,305; and 5,021,044; and PCT Publication WO92/11890. Catheters that provide for distal vessel access, as well asstents also can be used. Ultrasonically assisted drug delivery catheters(phonophoresis devices) are described in U.S. Pat. Nos. 5,362,309;5,318,014; and 5,315,998. Other iontophoresis and phonophoresis drugdelivery catheters are described in U.S. Pat. Nos. 5,304,120; 5,282,785;and 5,267,985. Sleeve catheters having drug delivery lumens intended foruse in combination with conventional angioplasty balloon catheters aredescribed in U.S. Pat. Nos. 5,364,356 and 5,336,178. All of thesereferences are herein incorporated by reference.

[0039] Direct intramuscular (IM) injections can be used to administerthe angiogenic agents of the invention. The agents for injection caninclude the FGF protein or angiogenically active fragments of theprotein as well as the gene or plasmid encoding the angiogenicallyactive FGF protein or fragment. Injections are administered to theaffected limb(s), in the thigh or calf, in the vicinity of existingvessels, near collateral flow vessels or conduit vessels such asarteries and arterials. The therapeutically effective dose of angiogenicagent is administered as a single injection, or can be divided andadministered as multiple injections. Preferably the therapeuticallyeffective amount or dose is delivered as 1 to about 20 injections, 1 toabout 15 injections, more preferably 1 to about 10 injections. A singledose of angiogenic agent can be administered intramuscularly, andrepeated as needed based on symptoms and/or hemodynamic measures. Localdelivery such as with IM injection can provide the added benefit ofadministering lower doses of the angiogenic agent. See Example 4 herein,and the copending application entitled “Dose of an Angiogenic Factor andMethod of Administering to Improve Myocardial Blood Flow,” filed Aug.11, 2000 and assigned U.S. patent application Ser. No. 09/637,471, basedon U.S. provisional application no. 60/148,746, filed Aug. 13, 1999,both of which herein incorporated by reference. The advantage to IMinjection(s) is that it is less likely to result in hypotension, is morelikely to have a longer half-life in the ischemic area, is lessinvasive, and therefore, can be repeated more frequently than the IAinfusion. An IA infusion or an IM injection(s) could be “boosted” by anIM injection(s) every 1-2 months as warranted by clinical symptoms.

[0040] Recombinant FGF-2 releases nitric oxide, a potent vasodilator,aggressive fluid management prior to (proactively) and during theinfusion is critical to patient's safety. Administration of IV fluids(e.g., 500-1000 mL of normal saline) to establish an estimated wedgepressure of 12 mm Hg prior to infusion and administration of boluses ofIV fluids (e.g., 200 mL normal saline) for decreases of systolic bloodpressure (e.g., <90 mm Hg) associated with infusion optimized the safetyof administration of rFGF-2 by IC or IV infusion to human patients.

[0041] Because a sudden bolus of rFGF-2 is associated with profoundhypotension in animals, the rate of infusion is critical to patient'ssafety. Administration at 0.5 to 2 mL per minute, typically 1 mL perminute, optimized the safety of administration of rFGF-2 by IC or IVinfusion to human patients.

[0042] In another embodiment of the invention, compositions comprisingfibroblast growth factor (FGF), including but not limited to FGF-2, canbe administered to a patient with peripheral artery disease, includingthose with claudication, in conjunction with vascular or mechanicalbypass surgery or angioplasty. The FGF, including but not limited toFGF-2, can be administered with and without a stent during surgery. TheFGF may thus be administered as an adjunct to vascular surgery involvingmechanical bypass and angioplasty.

[0043] The compositions of the invention provide a safe andtherapeutically effective amount of fibroblast growth factor to improveblood flow. By “safe and therapeutically effective amount” is intendedan amount of a fibroblast growth factor such as FGF-2, or angiogenicallyactive variant or fragment thereof, that when administered in accordancewith the invention is free from major complications that cannot bemedically managed, and that provides for objective improvement inpatients having symptoms of PAD. It is recognized that thetherapeutically effective amount may vary from patient to patientdepending upon age, weight, severity of symptoms, general health,physical condition, and the like. Other factors include the mode ofadministration and the respective amount of FGF included in thepharmaceutical composition. Typically, a therapeutically effectiveamount of an angiogenic agent of the invention, such as FGF-2, is about0.1 μg/kg to about 100 μg/kg, preferably about 0.20 μg/kg to about 75μg/kg, more preferably about 0.4 μg/kg to about 50 μg/kg, even morepreferably about 0.50 μg/kg to about 35 μg/kg, more preferably stillabout 1.0 μg/kg to about 30 μg/kg based on actual body weight. Thus,when the angiogenic agent is FGF-2, a therapeutically effective amountof FGF-2 is about 0. 1 μg/kg to about 1 μg/kg, 0.1 μg/kg to about 1μg/kg, about 1 μg/kg to 3 μg/kg, about 3 μg/kg to about 5 μg/kg, about 5μg/kg to about 7 μg/kg, about 7 μg/kg to about 8 μg/kg, about 8 μg/kg toabout 9 μg/kg, about 9 μg/kg to about 9.9 μg/kg, such as about 9.0, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, or 9.9 μg/kg, up to about 10 μg/kg,about 10 μg/kg to about 15 μg/kg, about 15 μg/kg to about 20 μg/kg,about 20 μg/kg to about 30 μg/kg, about 30 μg/kg to about 40 μg/kg,about 40 μg/kg to about 60 μg/kg, about 60 μg/kg to about 80 μg/kg ofthe rFGF-2, depending upon the route and the mode of administration.

[0044] As indicated, the compositions and methods of the invention areuseful for treating or preventing PAD and symptoms associated with PAD,including claudication and critical limb ischemia. In this manner, thedesired therapeutic responses include increased exercise capacity,improvement in ankle-brachial index, reduction in body pain andclaudication. In cases of PAD patients with critical limb ischemia,desired therapeutic responses include resolution of unremitting restpain that is not controllable by analgesic, healing of ulcers, andprevention of gangrene and amputation.

[0045] Methods for monitoring efficacy of administration of FGF,particularly FGF-2, for treatment of PAD are well known in the art. See,for example, methods for monitoring increased blood flow into affectedlimbs, including, but not limited to, Doppler ultrasound,plethysmography (Macdonald (1994) J. Vas. Tech. 18:241-248), andmagnetic resonance spectroscopy, ankle-brachial or toe systolic pressureindex at rest and after a period of exercise, and increased collateralvessel density using angiography. Clinical indicators of efficacyinclude total treadmill walk time (i.e., peak walking time, PWT) andtime to onset of claudication; and patient quality of lifequestionnaires.

[0046] The FGF-containing pharmaceutical compositions of the inventionwill be delivered for a time sufficient to achieve the desiredphysiological effect, i.e., angiogenesis, and/or restoration ofendothelial cell function and the promotion of collateral blood vessels.The compositions may be administered as a single bolus, or multipleinjections. Typically, the angiogenic factor will be delivered as aninfusion over a period of time. It is recognized that any means foradministration are encompassed including sustained-release formulations,plasmids, or genes, as well as other routes of administration. The totalamount of time may vary depending on the delivery rate and drugconcentration in the composition being delivered. For example, forintra-arterial administration, the time of administration may vary from1 second to about 24 hours, more usually from about 1 minute to about 6hours, specifically from about 5 minutes to about 30 minutes. A singleintra-arterial dose administration is efficacious in the treatment ofPAD.

[0047] When administered in accordance with the methods of theinvention, FGF-containing compositions provide the patient with a safeand therapeutically efficacious treatment for PAD that lasts at least 1month, 2 months, generally 3 months, 4 months, 6 months, and, in somecases, more than 6 months before a further treatment is needed. Theangiogenic agent, such as FGF-2, can be administered once or twice perday about every week, preferably every month or more preferably everyother month, even more preferably every 3 months, even more preferablyevery 4 months, and even more preferably still about every 6 months.

[0048] As indicated, fibroblast growth factors and related molecules areable to restore endothelial cell function and to promote endothelialand/or smooth muscle cell proliferation. The fibroblast growth factors(FGF) are a family of at least twenty-three structurally relatedpolypeptides (named FGF-1 to FGF-23) that are characterized by a highdegree of affinity for proteoglycans, such as heparin. The various FGFmolecules range in size from 15 to at least 32.5 kDa, and exhibit abroad range of biological activities in normal and malignant conditionsincluding nerve cell adhesion and differentiation (Schubert et al.(1987) J. Cell. Biol. 104:635-643); wound healing (U.S. Pat. No.5,439,818 (Fiddes)); as mitogens toward many mesodermal and ectodermalcell types, as trophic factors, as differentiation inducing orinhibiting factors (Elements et al. (1993) Oncogene 8:1311-1316); and asan angiogenic factor (Harada (1994) J. Clin. Invest. 94:623-630). Thus,the FGF family is a family of pluripotent growth factors that stimulateto varying extents fibroblasts, smooth muscle cells, epithelial cells,endothelial cells, myocytes, and neuronal cells. FGF-like polypeptidesare also contemplated for use in the compositions and methods of thepresent invention. By “FGF-like” is intended polypeptides that bind FGFreceptor 1, particularly receptor 1-C, bind to heparin-like molecules,and have angiogenic activity. By heparin-like molecule is intendedheparin, proteoglycans, and other polyanionic compounds that bind FGF,that dimerize FGF, and that facilitate receptor activation. Ofparticular interest in the practice of the invention is the FGFdesignated FGF-2 as well as variants and fragments thereof, which areknown in the art. For example, see U.S. Pat. Nos. 5,989,866; 5,925,528;5,874,254; 5,852,177; 5,817,485; 5,714,458; 5,656,458; 5,604,293;5,576,288; 5,514,566; 5,482,929; 5,464,943; and 5,439,818.

[0049] The FGF, more particularly FGF-2, to be administered can be fromany animal species including, but not limited to, avian, canine, bovine,porcine, equine, and human. Generally, the FGF is from a mammalianspecies, preferably bovine or human in the case of FGF-2. The FGF may bein the native, recombinantly produced, or chemically synthesized formsas outlined below. Where the FGF is FGF-2, it may be the 146 amino acidform, the 153-155 amino acid form, or a mixture thereof depending uponthe method of recombinant production. See U.S. Pat. No. 5,143,829,herein incorporated by reference. Further, angiogenically active muteinsof the FGF-2 molecule can be used. See, for example, U.S. Pat. Nos.5,859,208 and 5,852,177, herein incorporated by reference.

[0050] Biologically active variants of the FGF polypeptide of interest,more particularly FGF-2, are also encompassed by the methods of thepresent invention. As noted previously, such variants include fragments,analogues, and derivatives. Such variants should retain angiogenicactivities and thus be “angiogenically active” as measured usingstandard bioassays noted above.

[0051] Variants of the native FGF used in the compositions and methodsof the invention will generally have at least 70%, preferably at least80%, more preferably about 90% to 95% or more, and most preferably about98% or more amino acid sequence identity to the amino acid sequence ofthe reference FGF molecule. By “sequence identity” is intended the sameamino acid residues are found within the variant and the reference FGFmolecule when a specified, contiguous segment of the amino acid sequenceof the variant is aligned and compared to the amino acid sequence of thereference FGF molecule, which serves as the basis for comparison. Thus,for example, where the reference FGF-2 molecule is human FGF-2, anangiogenically active variant thereof will generally have at least 70%,preferably at least 80%, more preferably about 90% to 95% or more, mostpreferably about 98% or more, sequence identify to the full-length aminoacid sequence set forth in FIG. 3 (SEQ ID NO:3). In addition, other FGFreceptor-binding peptides can be used as described in, for example,WO98/21237 or U.S. application Ser. No. 09/407,687, filed Sep. 28, 1999,herein incorporated by reference.

[0052] A polypeptide that is a biologically active variant of areference polypeptide molecule of interest may differ from the referencemolecule by as few as 1-15 amino acids, as few as 1-10, such as 6-10, asfew as 5, as few as 4, 3, 2, or even 1 amino acid residue. Thepercentage sequence identity between two amino acid sequences iscalculated by determining the number of positions at which the identicalamino acid residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the segment undergoing comparison to thereference molecule, and multiplying the result by 100 to yield thepercentage of sequence identity.

[0053] For purposes of optimal alignment of the two sequences, thecontiguous segment of the amino acid sequence of the variant polypeptidemay have additional amino acid residues or deleted amino acid residueswith respect to the amino acid sequence of the reference polypeptidemolecule. The contiguous segment used for comparison to the referenceamino acid sequence will comprise at least twenty (20) contiguous aminoacid residues, and may be 30, 40, 50, 100, or more residues. Correctionsfor increased sequence identity associated with inclusion of gaps in thevariant's amino acid sequence can be made by assigning gap penalties.Methods of sequence alignment are well known in the art for both aminoacid sequences and for the nucleotide sequences encoding amino acidsequences.

[0054] Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm. Onepreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Myers and Miller (1988)CABIOS 4:11-17. Such an algorithm is utilized in the ALIGN program(version 2.0), which is part of the GCG sequence alignment softwarepackage. A PAM120 weight residue table, a gap length penalty of 12, anda gap penalty of 4 can be used with the ALIGN program when comparingamino acid sequences. Another preferred, nonlimiting example of amathematical algorithm for use in comparing two sequences is thealgorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLASTand XBLAST programs of Altschul et al. (1990)J. Mol. Biol. 215:403.BLAST nucleotide searches can be performed with the NBLAST program,score=100, wordlength=12, to obtain nucleotide sequences homologous to anucleotide sequence encoding the polypeptide of interest. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3, to obtain amino acid sequences homologous to thepolypeptide of interest. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be usedto perform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seewww.ncbi.nlm.nih.gov. Also see the ALIGN program (Dayhoff (1978) inAtlas of Protein Sequence and Structure 5:Suppl. 3 (National BiomedicalResearch Foundation, Washington, D.C.) and programs in the WisconsinSequence Analysis Package, Version 8 (available from Genetics ComputerGroup, Madison, Wisconsin), for example, the GAP program, where defaultparameters of the programs are utilized.

[0055] When considering percentage of amino acid sequence identity, someamino acid residue positions may differ as a result of conservativeamino acid substitutions, which do not affect properties of proteinfunction. In these instances, percent sequence identity may be adjustedupwards to account for the similarity in conservatively substitutedamino acids. Such adjustments are well known in the art. See, forexample, Myers and Miller (1988) Computer Applic. Biol. Sci. 4:11-17.

[0056] The art provides substantial guidance regarding the preparationand use of FGF polypeptide variants. In preparing the polypeptidevariants, one of skill in the art can readily determine whichmodifications to the native nucleotide or amino acid sequence willresult in a variant that is suitable for use as a therapeutically activecomponent of a pharmaceutical composition of the present invention foruse in the methods of the invention directed to treatment of patientshaving peripheral artery disease.

[0057] Fibroblast growth factors, such as FGF-2, are formulated intopharmaceutical compositions for use in the methods of the invention. Inthis manner, a pharmaceutically acceptable carrier may be used incombination with the angiogenic agent such as FGF-2 and other componentsin the pharmaceutical composition. By “pharmaceutically acceptablecarrier” is intended a carrier or diluent that is conventionally used inthe art to facilitate the storage, administration, and/or the desiredeffect of the therapeutic ingredients. A carrier may also reduce anyundesirable side effects of the angiogenic agent, i.e., FGF or variantthereof. A suitable carrier should be stable, i.e., incapable ofreacting with other ingredients in the formulation. It should notproduce significant local or systemic adverse effect in recipients atthe dosages and concentrations employed for therapy. Such carriers aregenerally known in the art. Suitable carriers for this invention arethose conventionally used large stable macromolecules such as albumin,gelatin, collagen, polysaccharide, monosaccarides, polyvinylpyrrolidone,polylactic acid, polyglycolic acid, polymeric amino acids, fixed oils,ethyl oleate, liposomes, glucose, sucrose, lactose, mannose, dextrose,dextran, cellulose, mannitol, sorbitol, polyethylene glycol (PEG),heparin alginate, and the like. Slow-release carriers, such ashyaluronic acid, may also be suitable. Stabilizers, such as trehalose,thioglycerol, and dithiothreitol (DTT), may also be added. See, forexample, copending U.S. Application Serial No. 60/229,238, entitled“Stabilized FGF Formulations Containing Reducing Agents,” hereinincorporated by reference. FGF formulations comprising DTT as describedin this application are defined herein as “stabilized FGF-DTTformulations and include stabilized FGF-2-DTT formatting.” Otheracceptable components in the composition include, but are not limitedto, buffers that enhance isotonicity such as water, saline, phosphate,citrate, succinate, acetic acid, and other organic acids or their salts.Further, the angiogenic agents of the invention may be administeredusing a patch for slow release. Such formulation may include DMSO.

[0058] Preferred pharmaceutical compositions may incorporate buffershaving reduced local pain and irritation resulting from injection. Suchbuffers include, but are not limited to, low phosphate buffers andsuccinate buffers. The pharmaceutical composition may additionallycomprise a solubilizing compound that is capable of enhancing thesolubility of an angiogenic agent or variant.

[0059] For the purposes of this invention, the pharmaceuticalcomposition comprising the angiogenic agent FGF or angiogenically activevariant thereof should be formulated in a unit dosage and in aninjectable or infusible form such as solution, suspension, or emulsion.It can also be in the form of lyophilized powder, which can be convertedinto solution, suspension, or emulsion before administration. Thepharmaceutical composition may be sterilized by membrane filtration,which also removes aggregates, and stored in unit-dose or multi-dosecontainers such as sealed vials or ampules.

[0060] The method for formulating a pharmaceutical composition isgenerally known in the art. A thorough discussion of formulation andselection of pharmaceutically acceptable carriers, stabilizers, andisomolytes can be found in Remington 's Pharmaceutical Sciences (₁₈thed.; Mack Pub. Co.: Eaton, Pa. 1990), herein incorporated by reference.

[0061] The pharmaceutical compositions of the present invention can alsobe formulated in a sustained-release form to prolong the presence of thepharmaceutically active agent in the treated patient, generally forlonger than one day. Many methods of preparation of a sustained-releaseformulation are known in the art and are disclosed in Remington 'sPharmaceutical Sciences (18^(th) ed.; Mack Pub. Co.: Eaton, Pa., 1990),herein incorporated by reference. Generally, the agent can be entrappedin semipermeable matrices of solid hydrophobic polymers. The matricescan be shaped into films or microcapsules. Examples of such matricesinclude, but are not limited to, polyesters, copolymers of L-glutamicacid and gamma ethyl-L-glutamate (Sidman et al. (1983) Biopolymers22:547-556), poly-actides (U.S. Pat. No. 3,773,919 and EP 58,481),polyactate polyglycolate (PLGA), hydrogels (see, for example, Langer etal. (1981)J. Biomed. Mater. Res. 15:167-277; Langer (1982) Chem. Tech.12:98-105), non-degradable ethylene-vinyl acetate, degradable lacticacid-glycolic acid copolymers such as the Lupron DepotTm, andpoly-D-(-)-3-hydroxybutyric acid (EP 133,988). Suitable microcapsulescan also include hydroxymethyl cellulose or gelatin-microcapsules andpoly-methylmethacylate microcapsules prepared by coacervation techniquesor by interfacial polymerization. Microparticles such as heparinalginate beads may also be used. In addition, microemulsions orcolloidal drug delivery systems such as liposomes and albuminmicrospheres, may also be used See a Remington 's PharmaceuticalSciences (₁₈th ed.; Mack Pub. Co.: Eaton, Pa., 1990).

[0062] In particular, a mammalian fibroblast growth factor of bovineorigin, FGF-2 of FIG. 2 (SEQ ID NO:2) also known as basic FGF (bFGF),and human FGF-2 of FIG. 3 (SEQ ID NO:4), or an angiogenically activefragment or mutein thereof, can be utilized in the practice of theinvention. The nucleotide sequence encoding bovine FGF-2 is set forth inFIG. 1 (SEQ ID NO: 1). The nucleotide sequence encoding human FGF-2 isset forth in FIG. 3 (SEQ ID NO:3). See also, U.S. Pat. No. 5,604,293,herein incorporated by reference. The dose of FGF-2 that is predicted toresult in clinical benefit to a patient whose exercise capacity islimited by claudication associated with PAD ranges from about 0.1 μg/kgto about 100 μg/kg of the FGF-2, preferably about 0.20 μg/kg to about 75μg/kg, more preferably about 0.4 μg,/kg to about 50 μg/kg, even morepreferably about 0.50 μg/kg to about 35 μg/kg, more preferably stillabout 1.0 μg/kg to about 30 μg/kg, and most likely from 0.3 to 3.5 mg asa standard dose. Thus, in one embodiment, the therapeutically effectivedose of FGF-2, such as recombinant FGF-2 (rFGF-2), is about 0.1 μg/kg toabout 1 μg/kg, about 1 μg/kg to 3 μg/kg, about 3 μg/kg to about 5 μg/kg,about 5 μg/kg to about 7 μg/kg, about 7 μg/kg to about 8 μg/kg, about 8Ag/kg to about 9 μg/kg, about 9 μg/kg to about 9.9 μg/kg, such as about9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, or 9.9 μg/kg, up to aboutμg/kg, about 10 μg/kg to about 15 μg/kg, about 15 μg/kg to about 20μg/kg, about 20 μg/kg to about 30 μg/kg, about 30 μg/kg to about 40μg/kg, about 40 μg/kg to about 60 μg/kg, about 60 μg/kg to about 80μg/kg of the FGF-2, depending on the route and mode of administration.

[0063] It is convenient to define the dose of angiogenic agent in moreabsolute terms that are not dependent upon the weight of the patient tobe treated. In this embodiment, the dose is referred to as a “standard”dose. When so defined, the standard dose to be administered inaccordance with the methods of the present invention ranges from about4.0 μg to about 7.2 mg, such as about 4.0 μg to about 0.3 mg, preferablyfrom about 0.3 mg to about 1.0 mg, even more preferably from about 1.0mg to about 2.0 mg, more preferably still from about 2.0 mg to about 2.5mg, from about 2.5 mg to 3.5 mg, from about 3.5 mg to about 4.5 mg, fromabout 4.5 mg to about 5.5 mg, from about 5.5 mg to about 6.5 mg, up toabout 7.2 mg. In this embodiment, the standard dose is a sufficientamount of FGF-2 to accommodate dosing any one of the majority of humanPAD patients, ranging from the smallest patient (e.g., 40 kg) at thelowest dosage (about 0.1 μg/kg) through the larger patients (e.g., 150kg) at higher dosages (about 48 μg/kg for this embodiment). For example,when a patient weighs 70 kg the standard dose ranges from about 0.2 mgto about 3.0 mg, from about 0.5 mg to about 2.5 mg, preferably about 2.1mg, depending upon the route and mode of administration.

[0064] Where lower doses of FGF-2 are contemplated, such as between 0.1μg/kg up to about 10 μg/kg, the standard dose to be administered inaccordance with the methods of the present invention ranges from about7.0 jig to about 0.7 mg, about 8 μg to about 0.6 mg, about 9 μg to about0.5 mg, about 0.1 mg to about 0.4 mg, preferably about 0.21 mg for a 70kg patient. Thus, in some embodiments, the standard dose for a 70 kgpatient ranges from about 7.0 μg to about 0.7 mg, including 8 μg, 9 μg,0.1 mg, 0.2 mg. 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.65 mg, up to about 0.7mg.

[0065] Because FGF-2 is a glycosaminoglycan- (e.g., heparin) bindingprotein and the presence of a glycosaminoglycan (also known as a“proteoglycan” or a “mucopolysaccharide”) optimizes activity and areaunder the curve (AUC), the dosages of FGF-2 of the present invention maybe administered within 20 to 30 minutes of an intravenous (IV)administration of a glycosaminoglycan, such as a heparin. Variousfractionated and unfractionated heparins, proteoglycans, and sulfatedmucopolysaccharides such as chondroitin sulfate can be used in thepractice of the invention. Low molecular weight heparins (<10,000 d) andunfractionated (i.e., high molecular weight) heparins (>10,000 d) can beused in the practice of the invention. These molecules can beadministered together with the rFGF-2 or within 20 to 30 minutes ofadministration of the rFGF-2. Heparin is suitably dosed at 20-80units/kg, and preferably at 40 units/kg.

[0066] In one embodiment, the unit dose contains a sufficient amount ofFGF-2 ranging from about 0.1 μg/kg to about 80 μg/kg. More typically,the systemic unit dose comprises 0.3 mg to 3.5 mg of the FGF-2 of FIG. 2(SEQ ID NO:2) or the FGF-2 of FIG. 3 (SEQ ID NO:4), or an angiogenicallyactive fragment or mutein thereof. Dosages for local delivery comprisingabout 0.01 μg to about 500 μg up to about 3 mg may be used. Whenadministered locally as with IM injections, the dose may be the same as,one-tenth of, or one-hundredth of the dose administeredintra-arterially. The unit dose is typically provided in solution orreconstituted lyophilized form containing the above-referenced amount ofFGF-2 and an effective amount of one or more pharmaceutically acceptablebuffers, stabilizers, and/or other excipients as described elsewhereherein.

[0067] The recombinant FGF-2 having the amino acid sequence of FIG. 2(SEQ ID NO:2) is made as described in U.S. Pat. No. 5,155,214, entitled“Basic Fibroblast Growth Factor,” which issued on Oct. 13, 1992, andwhich is incorporated herein by reference in its entirety. As disclosedin the '214 patent, a DNA of FIG. 1 (SEQ ID NO: 1), which encodes a bFGF(hereinafter “FGF-2”) of FIG. 2 (SEQ ID NO:2), is inserted into acloning vector, such as pBR322, pMB9, Col E 1, pCRI, RP4 or λ-phage, andthe cloning vector is used to transform either a eukaryotic orprokaryotic cell, wherein the transformed cell expresses the FGF-2. Inone embodiment, the host cell is a yeast cell, such as Saccharomycescerevisiae. The resulting full length FGF-2 that is expressed has 146amino acids in accordance with FIG. 2 (SEQ ID NO:2). Although the FGF-2of FIG. 2 (SEQ ID NO:2) has four cysteines, i.e., at residue positions25, 69, 87 and 92, there are no internal disulfide linkages. ['214 atcol. 6, lines 59-61.] However, in the event that cross-linking occurredunder oxidative conditions, it would likely occur between the residuesat positions 25 and 69.

[0068] The 146-residue mammalian FGF-2 of FIG. 2 (SEQ ID NO:2), which isof bovine origin, like the corresponding 146-residue human FGF-2 of FIG.3 (SEQ ID NO:4) is initially synthesized in vivo as a polypeptide having155 amino acids (Abraham et al. (1986) EMBO J. 5(10):2523-2528; FIG. 4(SEQ ID NO:6) of bovine origin; FIG. 5 (SEQ ID NO:8) of human origin).When compared to the full-length 155-residue FGF-2 molecules, the146-residue FGF-2 molecules lack the first nine amino acid residues,Met-Ala-Ala-Gly-Ser-Ile-Thr-Thr-Leu (SEQ ID NO:9), at the N-terminus ofthe corresponding full-length bovine and human 155-residue FGF-2molecules (FIG. 4 (SEQ ID NO:6) and FIG. 5 (SEQ ID NO:8), respectively).The 155-residueFGF-2 of human or bovine origin, and biologically activevariants thereof, can also be used in the compositions and methods ofthe present invention in the manner described for the bovine and human146-residue FGF-2 molecules. Again it is recognized that the 155-residueform may exist as 153-155 residues, or mixtures thereof, depending uponthe method of recombinant protein production. The mammalian FGF-2 ofFIG. 2 (SEQ ID NO:2) differs from human FGF-2 of FIG. 3 (SEQ ID NO:4) intwo residue positions. In particular, the amino acids at residuepositions 112 and 128 of the mammalian FGF-2 of FIG. 2 (SEQ ID NO:2) areSer and Pro, respectively, whereas in human FGF-2 (FIG. 3; SEQ ID NO:4),they are Thr and Ser, respectively. For the 155-residue forms, thesedifferences appear at residue positions 121 and 137 of FIG. 4 (SEQ IDNO:6; FGF-2 of bovine origin) and FIG. 5 (SEQ ID NO:8; FGF-2 of humanorigin).

[0069] The recombinant FGF-2 employed in the present compositions andmethods was purified to pharmaceutical quality (90% or greater purity byweight of total proteins, preferably 92% or greater purity, morepreferably 95% or greater purity, preferably substantially pure, that isabout 98% purity by weight of total proteins) using the techniquesdescribed in detail in U.S. Pat. No. 4,956,455, entitled “BovineFibroblast Growth Factor,” which issued on Sep. 11, 1990 and which isincorporated herein by reference in its entirety. In particular, thefirst two steps employed in the purification of the recombinant FGF-2used in a unit dose of a pharmaceutical composition of the invention are“conventional ion-exchange and HPLC purification steps as describedpreviously. “[U.S. Pat. No. 4,956,455, citing to Bolen et al. (1984)Proc. Natl. Acad. Sci. USA 81:5364-5368.I'm not sure about thesereferences.] The third step, which the '455 patent refers to as the “keypurification step” ['455 at col. 7, lines 5-6], is heparin-SEPHAROSE(®affinity chromatography, wherein the strong heparin binding affinity ofthe FGF-2 is utilized to achieve several thousand-fold purification wheneluting at approximately 1.4 M and 1.95 M NaCl ['455 at col. 9, lines20-25]. Polypeptide homogeneity may be confirmed by reverse-phase highpressure liquid chromatography (RP-HPLC). Buffer exchange was achievedby SEPHADEX® G-25(M) gel filtration chromatography.

[0070] In addition to the 146-residue FGF-2 of FIG. 2 (SEQ ID NO:2), thetherapeutically active agent in the unit dose of the present inventionalso comprises an angiogenically active fragment” of the FGF-2 of FIG. 2(SEQ ID NO:2). By the term “angiogenically active fragment of the FGF-2of FIG. 2 (SEQ ID NO:2)” is meant a fragment of FGF-2 that has about 80%of the 146 residues of FIG. 2 (SEQ ID NO:2) and that retains theangiogenic effect of the FGF-2 of FIG. 2 (SEQ ID NO:2). This definitionof “angiogenically active fragment” also applies to human FGF-2 of FIG.3 (SEQ ID NO:4). An “angiogenically active fragment” of the FGF-2 ofFIG. 4 (SEQ ID NO:6) or FIG. 5 (SEQ ID NO:8) is a fragment of FGF-2 thathas about 80% of the 155 residues of FIG. 4 (SEQ ID NO:6) or FIG. 5 (SEQID NO:8), respectively.

[0071] To be angiogenically active, the FGF-2 fragment should have twocell binding sites and at least one of the two heparin binding sites.The two putative cell binding sites of the analogous 146-residue humanFGF-2 (hFGF-2; SEQ ID NO:4) occur at about residue positions 36-39 andabout 77-81 thereof. See Yoshida et al. (1987) Proc. Natl. Aca. Sci. USA84:7305-7309, at FIG. 3. The two putative heparin binding sites ofhFGF-2 occur at about residue positions 18-22 and 107-111 thereof. SeeYoshida (1987), at FIG. 3. Given the substantial similarity between theamino acid sequences for human FGF-2 (hFGF-2) and bovine FGF-2 (bFGF-2),it is expected that the cell binding sites for bFGF-2 (FIG. 2 (SEQ IDNO:2)) are also at about residue positions 36-39 and about 77-81thereof, and that the heparin binding sites are at about residuepositions 18-22 and about 107-111 thereof. The additional 9 residues ofthe 155-residue form do not affect the relative positions of thesebinding sites with respect to residues 1-146 shown in FIG. 2 (SEQ IDNO:2; FGF-2 of bovine origin) or FIG. 3 (SEQ ID NO:4; FGF-2 of humanorigin). Thus, for the 155-residue form of human FGF-2 (FIG. 5; SEQ IDNO: 8), the two putative cell binding sites occur at about residuepositions 45-48 and about 86-90 thereof, and the two putative heparinbinding sites occur at about residue positions 27-31 and about 116-120thereof. Again, given the substantial similarity between the 155-residuebovine and human proteins, it is expected that the two putative cellbinding sites are at about residue positions 45-48 and about 86-90, andthe two putative heparin binding sites are at about residue positions27-31 and about 116-120 of the 155-residue bovine FGF-2 (FIG. 4; SEQ IDNO:6). Consistent with the above, it is well known in the art thatN-terminal truncations of the FGF-2 of FIG. 2 (SEQ ID NO:2) do noteliminate its angiogenic activity in cows. In particular, the artdiscloses several naturally occurring and biologically active fragmentsof the FGF-2 that have N-terminal truncations relative to the FGF-2 ofFIG. 2 (SEQ ID NO:2). An active and truncated bFGF-2 having residues12-146 of FIG. 2 (SEQ ID NO:2) was found in bovine liver and anotheractive and truncated bFGF-2, having residues 16-146 of FIG. 2 (SEQ IDNO:2) was found in the bovine kidney, adrenal glands, and testes. (SeeU.S. Pat. No. 5,155,214 at col. 6, lines 41-46, citing to Ueno et al.(1986) Biochem. Biophys. Res. Comm. 138:580-588.) Likewise, otherfragments of the bFGF-2 of FIG. 2 (SEQ ID NO:2) that are known to haveFGF activity are FGF-2 (24-120)-OH and FGF-2 (30-110)-N1712-[U.S. Pat.No. 5,155,214 at col. 6, lines 48-52.] These latter fragments retainboth of the cell binding portions of FGF-2 (FIG. 2 (SEQ ID NO:2)) andone of the heparin binding segments (residues 107-111). Accordingly, theangiogenically active fragments of a mammalian FGF typically encompassthose terminally truncated fragments of an FGF-2 that have at leastresidues that correspond to residues 30-110 of the FGF-2 of FIG. 2 (SEQID NO:2); more typically, at least residues that correspond to residues18-146 of the FGF-2 of FIG. 2 (SEQ ID NO:2).

[0072] It is recognized that other synthetic peptides based on nativeFGF sequences may be used as long as these peptides bind FGF receptors.Additionally hybrid FGF molecules may be constructed comprising peptidesfrom different native sequences as well as combinations of native andsynthetic sequences. Again, the hybrid molecules will retain the abilityto bind with FGF receptors.

[0073] The unit dose of the present invention also comprises an“angiogenically active mutein” of the FGF-2 of FIG. 2 (SEQ ID NO:2),FIG. 3 (SEQ ID NO:4), FIG. 4 (SEQ ID NO:6), or FIG. 5 (SEQ ID NO:8). Bythe term “angiogenically active mutein” is intended a mutated form ofthe FGF-2 of FIG. 2 (SEQ ID NO:2), FIG. 3 (SEQ ID NO:4), FIG. 4 (SEQ IDNO:6), or FIG. 5 (SEQ ID NO:8) that structurally retains at least 80%,preferably 90%, of the 146 residues of the FGF-2 sequence shown in FIG.2 (SEQ ID NO: 2), the 146 residues of the human FGF-2 sequence shown inFIG. 3 (SEQ ID NO:4), the 155 residues of the FGF-2 sequence shown inFIG. 4 (SEQ ID NO:6), or the 155 residues of the FGF-2 sequences shownin FIG. 5 (SEQ ID NO:8), respectively, in their respective positions,and that functionally retains the angiogenic activity of the respectiveunmutated form of FGF-2. Preferably, the mutations are “conservativesubstitutions” using L-amino acids, wherein one amino acid is replacedby another biologically similar amino acid. Examples of conservativesubstitutions include the substitution of one hydrophobic residue suchas Ile, Val, Leu, Pro, or Gly for another, or the substitution of onepolar residue for another, such as between Arg and Lys, between Glu andAsp, or between Gln and Asn, and the like. Generally, the charged aminoacids are considered interchangeable with one another. However, to makethe substitution more conservative, one takes into account both the sizeand the likeness of the charge, if any, on the side chain. Suitablesubstitutions include the substitution of serine for one or both of thecysteines at residue positions 87 and 92, which are not involved indisulfide formation. Other suitable substitutions include anysubstitution wherein at least one constituent cysteine is replaced byanother amino acid so that the mutein has greater stability under acidicconditions, see for example U.S. Pat. No. 5,852,177 which is hereinincorporated by reference. One such substitution is the replacement ofcysteine residues with neutral amino acids such as for example: glycine,valine, alanine, leucine, isoleucine, tyrosine, phenylalanine,histidine, tryptophan, serine, threonine, and methionine (U.S. Pat. No.5,852,177). Preferably, substitutions are introduced at the FGF-2N-terminus, which is not associated with angiogenic activity. However,as discussed above, conservative substitutions are suitable forintroduction throughout the molecule.

[0074] One skilled in the art, using well-known techniques, is able tomake one or more point mutations in the DNA of FIG. 1 (SEQ ID NO:1),FIG. 3 (SEQ ID NO:3), FIG. 4 (SEQ ID NO:5), or FIG. 5 (SEQ ID NO:7) toobtain expression of an FGF-2 polypeptide mutein (or fragment of amutein) having angiogenic activity for use within the unit dose,compositions, and methods of the present invention. To prepare anangiogenically active mutein of the FGF-2 of FIG. 2 (SEQ ID NO:2), FIG.3 (SEQ ID NO:4), FIG. 4 (SEQ ID NO:6), or FIG. 5 (SEQ ID NO:8), one usesstandard techniques for site-directed mutagenesis, as known in the artand/or as taught in Gilman et al. (1979) Gene 8:81 or Roberts et al.(1987) Nature 328:73 1, to introduce one or more point mutations intothe cDNA of FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) FIG. 4 (SEQ IDNO:5), or FIG. 5 (SEQ ID NO:7) that encodes the FGF-2 of FIG. 2 (SEQ IDNO:2), FIG. 3 (SEQ ID NO:4), FIG. 4 (SEQ ID NO:6), or FIG. 5 (SEQ IDNO:8), respectively.

[0075] Pharmaceutical compositions of the invention comprise anangiogenically effective dose of a mammalian FGF-2 of FIG. 2 (SEQ IDNO:2), FIG. 3 (SEQ ID NO:4), FIG. 4 (SEQ ID NO:6), FIG. 5 (SEQ ID NO:8)or an angiogenically active fragment or mutein thereof, and apharmaceutically acceptable carrier. Typically, the safe andangiogenically effective dose of the pharmaceutical composition of thepresent invention is in a form and a size suitable for administration toa human patient and comprises (i) 1.0 μg/kg to 30.0 μg/kg of an FGF-2 ofFIG. 2 (SEQ ID NO:2) or an angiogenically active fragment or muteinthereof, (ii) and a pharmaceutically acceptable carrier. In otherembodiments, the safe and angiogenically effective dose comprises about0.1 μg/kg to about 1 μg/kg, about 1 μg/kg to 3 μg/kg, about 3 μg/kg toabout 5 μg/kg, about 5 μg/kg to about 7 μg/kg, about 7 μg/kg to about 8μg/kg, about 8 μg/kg to about 9 μg/kg, about 9 μg/kg to about 9.9 μg/kg,such as about 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, or 9.9 μg/kg,up to about 10 μg/kg, about 10 μg/kg to about 15 μg/kg, about 15 μg/kgto about 20 μg/kg, about 20 μg/kg to about 30 μg/kg, about 30 μg/kg toabout 40 μg/kg, about 40 μg/kg to about 60 μg/kg, about 60 μg/kg toabout 80 μg/kg of the FGF-2 of FIG. 2 (SEQ ID NO:2), FIG. 3 (SEQ IDNO:4), FIG. 4 (SEQ ID NO:6), FIG. 5 (SEQ ID NO:8) or an angiogenicallyactive fragment or mutein thereof, and a pharmaceutically acceptablecarrier.

[0076] A typical pharmaceutical composition comprises 0.1 mg/ml to 10mg/ml, more typically 0.3 mg/ml to 0.5 mg/ml, of FGF-2, moreparticularly recombinant FGF-2 (rFGF-2), having the sequence set forthin FIG. 2 (SEQ ID NO:2), or in FIG. 3 (SEQ ID NO:4), or anangiogenically active fragment or mutein thereof, 10 mM thioglycerol,135 mM NaCl, 10 mM Na citrate, and 1 mM EDTA, pH 5.0. A suitable diluentor flushing agent for the above-described composition is any of theabove-described carriers. Typically, the diluent is the carrier solutionitself comprising 10 mM thioglycerol, 135 mM NaCl, 10 mM Na citrate, and1 mM EDTA, pH 5.0. The rFGF-2 of FIG. 2 (SEQ ID NO:2) or anangiogenically active fragment or mutein thereof is unstable for longperiods of time in liquid form. To maximize stability and shelf life,the pharmaceutical composition of the present invention comprising aneffective amount of rFGF-2 or an angiogenically fragment or muteinthereof, in a pharmaceutically acceptable aqueous carrier should bestored frozen at −60° C. Thawed, the solution is stable for 1 month atrefrigerated conditions. A typical unit dose would comprise about 5-10ml of the above described composition having 1.5-8 mg of FGF-2 of FIG. 2(SEQ ID NO:2), or FIG. 3 (SEQ ID NO:4).

[0077] In another embodiment, the pharmaceutical composition comprises aunit dose of FGF-2 of FIG. 2 (SEQ ID NO:2), FIG. 3 (SEQ ID NO:4), or anangiogenically active fragment or mutein thereof in lyophilized(freeze-dried) form. In this form, the unit dose of FGF-2 would becapable of being stored at room temperature for substantially longerthan 6 months without loss of therapeutic effectiveness. Lyophilizationis accomplished by the rapid freeze drying under reduced pressure of aplurality of vials, each containing a unit dose of the FGF-2 of thepresent invention therein. Lyophilizers, which perform the abovedescribed lyophilization, are commercially available and readilyoperable by those skilled in the art. Prior to administration to apatient, the lyophilized product is reconstituted to a knownconcentration, preferably in its own vial, with an appropriate sterileaqueous diluent, typically 0.9% (or less) sterile saline solution, or acompatible sterile buffer, or even sterile deionized water. See, forexample, copending U.S. Application Serial No. 60/229,238, entitled“Stabilized FGF Formulations containing Reducing Agents,” hereinincorporated by reference. Depending upon the weight of the patient inkg, a single dose comprising from 0.2 μg/kg to 36 μg/kg of the FGF-2 ofFIG. 2 (SEQ ID NO:2), the FGF-2 of FIG. 3 (SEQ ID NO:4), or anangiogenically active fragment or mutein thereof is withdrawn from thevial as reconstituted product for administration to the patient. Forexample, an average 70 kg man that is being dosed at 24 μg/kg, wouldhave a sufficient volume of the reconstituted product withdrawn from thevial to receive an infusion of (70 kg x 24 μg/kg) 1680 μg (i.e., 1.680mg).

[0078] The pharmaceutical composition in solution form is generallyadministered by infusing the unit dose substantially continuously over aperiod of about 10 to about 30 minutes, although it is recognized thatthe composition may be administered over a longer period of time. Whenthe composition is administered into more than one blood vessel,typically, a portion (e.g., one half) of the unit dose is administeredin a first vessel followed by administration into a second secondaryvessel. Using the above-described repositioning procedure, portions ofthe unit dose may be administered to a plurality of vessels until theentire unit dose has been administered. After administration, thecatheter is withdrawn using conventional protocols known in the art.Signs of angiogenesis and a therapeutic benefit, such as reducedclaudication, improvement in ankle-brachial index, improvement in peakwalking time, increase in ability to climb stairs, reduced body pain,improvement in or prevention of critical limb ischemia, and improvedpatient quality of life are seen as early as two weeks to one monthfollowing the FGF-2 administration.

[0079] The following examples are offered by way of illustration and notby way of limitation.

EXPERIMENTAL Example 1 Unit Dose of rFGF-2 Employed in a Phase IClinical Trial

[0080] The recombinantly produced FGF-2 (rFGF-2) having the sequenceshown in FIG. 2 (SEQ ID NO:2) was formulated as a unit dose andpharmaceutical composition. The various formulations are describedbelow.

[0081] The rFGF-2 unit dose was provided as a liquid in 3 cc type Iglass vials with a laminated gray butyl rubber stopper and red flip-offoverseal. The rFGF-2 unit dose contained 1.2 ml of 0.3 mg/ml rFGF-2 ofFIG. 2 (SEQ ID NO:2) in 10 mM sodium citrate, 10 mM monothioglycerol, 1mM disodium dihydrate EDTA (molecular weight 372.2), 135 mM sodiumchloride, pH 5.0. Thus, in absolute terms, each vial (and unit dose)contained 0.36 mg rFGF-2. The vials containing the unit dose in liquidform were stored at 2° to 8° C.

[0082] The diluent was supplied in 5 cc type I glass vials with alaminated gray butyl rubber stopper and red flip-off overseal. TherFGF-2 diluent contains 10 mM sodium citrate, 10 mM monothioglycerol,135 mM sodium chloride, pH 5.0. Each vial contained 5.2 ml of rFGF-2diluent solution that was stored at 2 to 8 C. Such agents may also beadministered to prevent progression of critical limb ischemia toamputation.

[0083] The rFGF-2 pharmaceutical composition that was infused wasprepared by diluting the rFGF-2 unit dose with the rFGF diluent. Inorder to keep the EDTA concentration below the limit of 100 μg/ml, thetotal infusion volume was increased up to 40 ml when proportionatelyhigher absolute amounts of FGF-2 were administered to patients.

Example 2 Phase II PAD Clinical Trial

[0084] Peripheral artery disease (PAD), as defined by restinganklebrachial index (ABI) less than 0.9, is a common conditionafflicting about 15% of adults greater than 55 years of age. About 33%of these individuals are symptomatic with claudication; about 25% willprogress. With worsening blood flow limitation, the spectrum of PAD runsfrom mild to moderate to severe claudication, followed bylimb-threatening ischemia, initially characterized by rest pain, thenpoor wound healing, and impending or overt gangrene.

[0085] A phase II trial was undertaken to assess the efficacy ofintra-arterial administration of rFGF-2 on exercise capacity in patientswith intermittent claudication due to infra-inguinal PAD. The phase IIPAD trial was a multicenter, randomized, double-blind,placebo-controlled, regimen finding study of rFGF-2 to evaluate thesafety, pharmocokinetics, and efficacy by intra-arterial (IA) infusionover 20 minutes in PAD subjects with moderate to severe intermittentclaudication. Major selection criteria for inclusion of a patient in thetrial were age greater than 40 years, exercise limited by claudication,index ankle brachial index (ABI) of less than 0.8 at rest, patentfemoral inflow, medically stable for greater than 4 months, and informedconsent. Major selection criteria for exclusion of a patient from thetrial were evidence of malignancy (according to ACS guidelines),creatinine greater than 2.0 mg/dL, urine protein greater than or equalto 2+or greater than 300 mg/day, proliferative retinopathy, and/or otherconditions impacting safety or compliance. 190 patients participated inthe phase II PAD trial. Baseline characteristics of the patientpopulation are shown in Tables 1-3. TABLE 1 Baseline characteristics ofthe phase II PAD clinical trial patient population. Placebo SINGLEDOUBLE Any FGF Number of Subjects 63 66 61 127 Median Age (yrs) 67 65 6867 Male 73% 71% 82% 76% Female 27% 29% 18% 24% ABI at Rest (index) 0.550.57 0.55 0.56 PWT at Baseline 5.32 5.15 5.81 5.48 COT at Baseline 1.972.03 2.20 2.13 Current Smoker 38% 24% 21% 23% Past Smoker 43% 59% 61%60% Never Smoker 19% 17% 18% 17% Structured Exercise 56% 50% 49% 50%

[0086] TABLE 2 Concurrent diagnoses of the target patient population inthe phase II PAD clinical trial. Placebo SINGLE DOUBLE Any FGF CARDIACHistory of CAD 62% 58% 62% 60% History of CHF 8% 14% 11% 13% Previous MI29% 30% 31% 30% CAD Angioplasty (PTCA) 21% 27% 21% 24% S/P CABG 41% 33%31% 32% Prior PAD Surgery 48% 44% 56% 50% Prior PTI 21% 27% 21% 24%CEREBROVASCULAR Previous Stroke 8% 6% 7% 6% RISK FACTORS DiabetesMellitus 36% 27% 38% 33% Hyperlipidemia 75% 77% 75% 76% Hypertension 79%67% 75% 71%

[0087] TABLE 3 Incidence of peripheral angioplasty and limbrevascularization in the target population of the phase II PAD clinicaltrial. Indicators of quality of life as measured by WIQ and SF-36 arealso shown. Placebo SINGLE DOUBLE Any FGF Peripheral Angioplasty None71% 71% 71% 71% One  7% 14% 17% 16% >one 22% 14% 12% 13% LimbRevascularization None 72% 68% 66% 67% One 16% 16% 17% 17% >one 12% 16%17% 17% WIQ - distance score 14% 18% 23% 21% WIQ - speed score 19% 21%26% 23% WIQ - stair climbing score 32% 23% 37% 30% SF-36 - PCSS 30.329.9 32.9 31.6

[0088] Approximately two-thirds of the patients had a history ofcoronary artery disease (CAD), slightly less than one-third hadexperienced myocardial infarction, one-third were diabetic,approximately three-fourths had hypertension, and/or dyslipidemia (Table2). Approximately 20-30% of this target population had undergone greaterthan one vascularization procedure (Table 3). The low baseline qualityof life scores (WIQ and SF-36) are indicative of a target PAD patientpopulation with moderate to severe disease. The scores are based on ascale where 1 or 100% is normal. Thus an increase in the scorerepresents an improvement. Scores are tabulated based on a questionnairewhere patients perform a self-evaluation.

[0089] The rFGF-2 was administered by intra-arterial (IA) infusion over20 minutes divided between two legs on days 1 and 30. The doseadministered was 30 μg/kg of rFGF-2. The trial patients were dividedinto three groups: placebo; single dose (rFGF-2 on day 1); and doubledose (rFGF-2 on days 1 and 30). The primary endpoint used in the studywas a change in peak walking time (PWT) at day 90 on a Gardner gradedexercise protocol. Secondary endpoints measured included: change in PWTat day 180, claudication onset time (COT; noted as the time at which thepatient indicates claudication and/or pain begins), ankle-brachialpressure index (ABI; as determined using standard ultrasound device),and health-related quality of life (QOL) by Walking ImpairmentQuestionnaire (WIQ) and Short-Form-36 (SF-36) at day 90 and day 180.

[0090] Recombinant FGF-2 (rFGF-2) was formulated in a solutioncontaining 0.3 mg/ml rFGF-2, 10 mM sodium citrate, 0.3 mM EDTA, 10 mMthioglycerol, 135 mM sodium chloride, pH 5.0. Each 5 ml vial contained3.7 ml of clear colorless solution (1. 1 mg rFGF-2 per vial). Vialscontaining rFGF-2 were labeled “rFGF-2” and supplied frozen. Drugproduct was thawed at room temperature prior to preparation of dose;detailed instruction for pharmacists were provided in study manuals.Thawed, undiluted active drug product could be stored refrigerated at2-8° C. for 30 days.

[0091] Drug product was diluted with placebo (diluent) and filteredbefore administration. The filter was sterile, non-pyrogenic, and lowprotein binding. Filtration of the drug product through a 0.22 micronsyringe filter (e.g., Millipore, Millex-GV, #SLGVR25LS or equivalent)would remove particle with no resultant loss in strength or potency.Thawed, undiluted drug product was used within 8 hours.

[0092] Placebo (diulent) was supplied as a clear, colorless solutionindistinguishable from the drug product. It contained 10 mM sodiumcitrate, 0.3 mM EDTA, 10 mM thioglycerol, 135 mM sodium chloride, pH5.0. Vials containing diluent were labeled “placebo,” supplied in aliquid state, and stored refrigerated at 2-8° C.

[0093] The results of the trial indicated that rFGF-2 had an acceptablesafety profile at 90 days for both the single- and double-dose treatmentgroups. Dosing at day 1 and day 30 yielded similar safety data as singledosing at day 1 (data not shown).

[0094] Patient disposition and adverse events for patients at day 180 ofthe study are shown in Tables 4 and 5, respectively. TABLE 4 Patientfollow-up. Placebo SINGLE DOUBLE Randomized: 63 66 61 Safety: 180 day FU57 63 56 PWT: 90/180 days 58/54 62/61 54/53 Premature Termination 6 3 5Death 1 0 1 Adverse Event 1 0 0 Withdrew Consent 2 1 2 Lost to FU 2 2 2Revascularized/Amputation 3 2 3

[0095] TABLE 5 Safety: Adverse Events Placebo SINGLE DOUBLE Any FGFNumber of Subjects 63 66 61 127 Any AE 41 (65%) 43 (65%) 46 (75%) AnyCardiac AE 8 6 7 13 Hypotension 2 4 5 9 Proteinuria 2 6 7 13 Serious AEs13 (21%)  9 (14%) 14 (23%) Deaths 1 0 1 1 Serious Cardiac AEs 3 4 2 6Revascularizations/ 3 2 3 5 Amputations Gangrene (2) 0 0 0 Malignancy 10 0 0 Retinal Disorders 1 1 0 1 Pleural/Pericar- 1 0 0 0 dial Effusion

[0096] Data Analysis

[0097] Primary analysis of the data was performed by ANOVA. Ten subjectswith missing PWT and 6 subjects who were revascularized were excludedfrom the analysis. Secondary analysis was performed by ANOVA of Ranks.The 16 subjects excluded from the primary analysis were assigned lowestrank. This represents a more conservative approach. See, for example,Table 6. TABLE 6 Evaluable vs intent to treat analysis. Hypotheticalsubjects Baseline PWT Day 90 PWT ANOVA Ranks n = 6 n = 4 n = 6 a 8:4010:20 1:40 4 b 5:30 6:55 1:25 3 c 4:30 5:00 → 0:30 2 d 6:20 6:00 −0:20 1e 4:00 Angioplasty day 60 1 f 2:30 Surgery day 89 1

[0098] Primary Endpoint: Peak Walking Time at Day 90

[0099] Recombinant FGF-2 was efficacious at treating PAD as measured bya statistically significant improvement in the PWT (p=0.026) at day 90in the patients in the trial receiving a single dose of rFGF-2 comparedto the control placebo group (FIG. 6). Also, results indicated that adouble dose of rFGF-2 (days 1 and 30) was not better than a single dose(day 1).

[0100] Secondary Efficacy Variables

[0101] Secondary efficacy variables included PWT at day 180,claudication onset time (COT) and ankle brachial index (ABI) at days 90and 180, and WIQ and SF-36 quality of life questionnaires at days 90 and180. Results at day 180 reflect a large increase in placebo response.

[0102]FIG. 7 shows absolute change in PWT at days 90 and 180 for thepatient groups receiving placebo, single-dose rFGF-2, or double-doserFGF-2. For each patient the PWT at baseline is subtracted from the PWTat day 90 and the differences are summed for each group and a meandetermined. The data are analyzed by an analysis of variance (ANOVA).

[0103]FIG. 8 shows the percent absolute change in PWT in the threepatient groups shown at day 90 and day 180. The percent change in PWTaveraged across the two rFGF-2 groups is also shown (designated AnyFGF).

[0104]FIG. 9 shows the measured ABI (ankle brachial index) for the threepatient groups of the phase II clinical study. A baseline measurement, aday-90 measurement, and the corresponding change between the baselineand day-90 measurement are indicated. The mean change in ABI is alsoshown for the three patient groups. The ABI is described in An OfficeBased Approach to the Diagnosis and Treatment of Peripheral ArterialDisease (2000) Society of Vascular Medicine and Biology (MedicalCommunications Media, Inc., Wrightstown, Pa.).

[0105] The anklebrachial index is the ratio of the systolic pressure inthe foot to the systolic pressure in the arm as measured by a Dopplerultrasound device. The normal ABI is 1. An ABI less than 0.9 isconsidered diagnostic of PAD. The mean ABI of the target populationenrolled in the trial was .56 in the index leg at rest. The index leg isthe leg with the lower ABI. FIG. 9 shows the mean ABI (top panel) atbaseline, day 90, and day 180 for each group. The bottom panel shows themean change in ABI at day 90 and at day 180 for each group. There is apositive directional change in the treatment groups compared to placebo.The difference achieved statistical significance in the double-dosegroup at day 180 (mean ΔABI=0.11; p=0.031 versus placebo). As the ABIrepresents an objective measure of blood flow, this change is consistentwith a proposed mechanism of FGF, the formation of new collateral bloodvessels.

[0106]FIG. 10 represents the WIQ severity of claudication at days 90 and180 for single- and double-dose groups relative to the placebo group.The bar values represent the percentage of patients in each group whoimproved, stayed the same, or became worse in each group. At day 90,greater than 50% of the patients in the treatment groups were improvedwhereas less than 40% of the placebo patients were improved. At day 180this apparent treatment benefit is lost. For further information on theWIQ, see Regensteiner et al. (1990) J. Vasc. Med. Biol. 2:142-153 hereinincorporated by reference.

[0107]FIG. 11 shows the severity scores at baseline, day 90, and day 180for distance, speed, and stair climbing for each group. While changesare directionally positive for the FGF treatment groups for distance andspeed, the results did not achieve statistical significance. For stairclimbing, there was a trend of improvement for the single-dose groupversus the placebo group (p=.11). The figure shows that results for thesingle-dose group were better than results for placebo group for WIQdistance, speed, and stair climbing. The figure is shown with a scalewhere higher scores are better.

[0108]FIG. 12 depicts the physical summary scores from the short form 36(SF-36). The SF-36 is a general validated quality of life instrumentconsisting of 36 questions. The SF-36 has 12 domains, which can becollapsed into two summary scores, physical and mental. A change of 1point is associated with an increased lifespan of 2 years. The changescores in the figure indicate an improvement in the single-dose groupversus the placebo group by greater than 2 points at day 90.

[0109] Results of the study are summarized in FIG. 13.

[0110] Examination of Subgroups

[0111] The analysis plan provided for the tracking of treatment responsein three pre-specified subgroups of the phase II clinical trial patientpopulation: diabetes (type I or II, yes vs no), smoking (current vsnon-current, which included those individuals who had smoked in the pastor had never smoked), and median age (<68 years vs >68 years). Theresults of the primary efficacy measure, change in PWT, are presentedbelow for each subgroup. Data in Tables 7-12 reflect the log-transformedresults (to be consistent with the primary efficacy analysis).

[0112] Diabetes

[0113] The results of PWT at days 90 and 180 are presented in Tables 7and 8. There was no statistically significant difference between theplacebo and FGF-treated groups. Changes were greater in both diabeticsand non-diabetics in the FGF-treated groups at day 90. At day 180,changes were smaller in diabetics and similar in non-diabetics in theFGF-treated groups. The percentage of diabetics in the single-dose groupwas slightly lower (27% versus 37% in the placebo group, and 36% in thedouble-dose group). TABLE 7 Change in PWT at day 90 and day 180 indiabetics. Placebo Single Double Any FGF Day 90 N = 21 N = 17 N = 22 N =39 Relative Change in PWT 12.3% 26.8% 18.8% 21.9% Pair-wise P-value .44.70 .53 Mean Change in PWT 1.08 1.42 1.26 1.33 (min) (+/− SD) (1.84)(2.74) (3.08) (2.90) Overall P value = .74 Day 180 N = 20 N = 17 N = 21N = 38 Relative Change in PWT 18.2% 7.6% 4.6% 5.8% Pair-wise P-value .64.52 .52 Mean Change in PWT 1.85 1.32 1.07 1.18 (min) (+/31 SD) (2.47)(3.27) (2.94) (3.05) Overall P value = .81

[0114] TABLE 8 Change in PWT at day 90 and day 180 in non-diabetics.Placebo Single Double Any FGF Day 90 N = 37 N = 45 N = 32 N = 77Relative Change in PWT 13.0% 34.9% 18.7% 28.0 Pair-wise P-value .061 .65.14 Mean Change in PWT .75 2.12 1.90 2.03 (min) (+/− SD) (2.60) (2.88)(3.61) (3.18) Overall P value = .14 Day 180 N = 34 N = 44 N = 32 N = 76Relative Change in PWT 19.1% 22.7% 20.0% 21.6% Pair-wise P-value .80 .96.84 Mean Change in PWT 1.57 2.15 1.89 2.04 (min) (+/− SD) (2.91) (3.97)(2.89) (3.54) Overall P value = .96

[0115] Smoking

[0116] The results of the change in PWT at days 90 and 180 for currentand non-current smokers are presented in Tables 9 and 10. For currentsmokers, no statistically significant difference was seen at day 90 orday 180. For non-current smokers at day 90,there was a statisticallysignificant difference overall (p=0.007) and between the placebo andsingle-dose groups (P-value=.002); at day 180, no statisticallysignificant difference was seen. The results of regression analysissuggested smoking was a confounder of outcome. TABLE 9 Change in PWT atday 90 and day 180 in current smokers. Placebo Single Double Any FGF Day90 N = 23 N = 14 N = 13 N = 25 Relative Change in PWT 29.3% 22.1% 31.3%26.2% Pair-wise P-value .76 .93 .87 Mean Change in PWT 1.55 2.27 2.362.31 (min) (+/− SD) (2.67) (4.22) (3.91) (4.00) Overall P value = .93Day 180 N= 20 N = 14 N = 11 N = 25 Relative Change in PWT 27.2% 1.0%43.3% 18.1% Pair-wise P-value .25 .53 .66 Mean Change in PWT 2.03 1.892.19 2.02 (min) (+/− SD) (2.45) (4.55) (3.24) (3.95) Overall P value =.26

[0117] TABLE 10 Change in PWT at day 90 and day 180 in non-currentsmokers. Placebo Single Double Any FGF Day 90 N = 35 N = 48 N = 43 N =91 Relative Change in PWT 5.5% 35.9% 15.8% 26.1% Pair-wise P-value .002.27 .019 Mean Change in PWT 0.42 1.83 1.45 1.65 (min) (+/− SD) (2.02)(2.34) (3.27) (2.81) Overall P value = .007 Day 180 N = 34 N = 47 N = 42N = 89 Relative Change in PWT 19.8% 29.9 10.0% 20.3% Pair-wise P-value.46 .46 .97 Mean Change in PWT 1.46 1.93 1.40 1.68 (min) (+/− SD) (2.90)(3.57) (2.84) (3.24) Overall P value = .28

[0118] Age

[0119] The results of the change in PWT at days 90 and 180 forsubjects>68 years of age and ≦68 years of age are presented in Tables 11and 12. There was a greater frequency of subjects with high PWT (≧8minutes) in the double-dose group. For subjects >68 years, anunfavorable statistically significant difference was seen in thedouble-dose group at day 180 (P-value=0.031). For subjects <68 years,the change in PWT was higher in the FGF-treated groups, but nostatistically significant difference was seen at day 90 or day 180. Theresults suggest a greater improvement in subjects ≦68 years of age.TABLE 11 Change in PWT at day 90 and day 180 in subjects >68 years ofage. Placebo Single Double Any FGF Day 90 N = 27 N = 24 N = 30 N = 54Relative Change in PWT 16.0% 29.1% 8.0% 17.6% Pair-wise P-value .27 .53.87 Mean Change in PWT 1.00 1.58 0.81 1.15 (min) (+/−SD) (2.18) (2.66)(2.25) (2.45) Overall P value = .22 Day 180 N = 25 N = 25 N = 29 N = 54Relative Change in PWT 28.8% 7.1% −4.0% 1.4% Pair-wise P-value .19 .031.044 Mean Change in PWT 1.74 0.86 1.05 0.96 (min) (+/−SD) (2.73) (2.89)(2.75) (2.79) Overall P value = .095

[0120] TABLE 12 Change in PWT at day 90 and day 180 in subjects ≦68years of age. Placebo Single Double Any FGF Day 90 N = 31 N = 38 N = 24N = 62 Relative Change in PWT 15.0% 33.1% 40.0% 35.7% Pair-wise P-value.18 .11 .099 Mean Change in PWT 0.76 2.16 2.67 2.36 (min) (+/−SD) (2.51)(2.96) (4.25) (3.49) Overall P value = .23 Day 180 N = 29 N = 36 N = 24N = 60 Relative Change in PWT 25.8% 31.5% 48.2% 37.9% Pair-wise P-value.72 .25 .44 Mean Change in PWT 1.61 2.65 2.19 2.47 (min) (+/−SD) (2.78)(4.17) (3.03) (3.73) Overall P value = .50

[0121] Post-Hoc Responder Analysis

[0122] Of those subjects in the study whose PWT increased by ≧2 minutesat day 90,there was a higher frequency of subjects with low PWT (≦4minutes) at baseline in the single-dose group. The strongest predictorof response at day 180 was the response at day 90. The change in PWT forsubjects whose PWT increased by ≧2 minutes and for subjects whose PWTincreased by <2 minutes are presented in Tables 13 and 14, respectively.There was a higher percentage of responders at days 90 and 180 and themagnitude of the change was greater in the single-dose group. About 40%of the patients receiving the single-dose treatment had an increase inPWT of greater than 2 minutes at both day 90 and day 180, while onlyabout 22% and about 26% of the patients receiving placebo or a doubledose of FGF-2 experienced this magnitude of response. In addition, thetreatment effect appears to persist at day 180 by this analysis. TABLE13 Change in PWT at day 90 and day 180 in subjects whose PWT increasedby ≧2 minutes at day 90. Placebo Single Double Any FGF Day 90 N = 14 N =27 N = 16 N = 43 Relative Change in PWT 62.1% 85.8% 68.4% 79.0%Pair-wise P-value .17 .76 .27 Mean Change in PWT 3.67 4.28 5.37 4.68(min) (+/−SD) (1.57) (2.54) (4.10) (3.20) Overall P value = .35 Day 180N = 14 N = 26 N = 16 N = 42 Relative Change in PWT 55.3% 86.0% 53.2%72.7% Pair-wise P-value .17 .90 .37 Mean Change in PWT 3.02 4.28 4.164.24 (min) (+/−SD) (2.39) (3.85) (3.31) (3.61) Overall P value = .24

[0123] TABLE 14 Change in PWT at day 90 and day 180 in subjects whosePWT increased by <2 minutes at day 90. Placebo Single Double Any FGF Day90 N = 44 N = 35 N = 38 N = 73 Relative Change in PWT 3.8% 4.4% 3.9%4.1% Pair-wise P-value .93 .99 .95 Mean Change in PWT −.02 .12 .07 .09(min) (+/−SD) (1.78) (1.33) (1.04) (1.18) Overall P value = 1.00 Day 180N = 40 N = 35 N = 37 N = 72 Relative Change in PWT 14.5% −5.8% 2.5%−1.4% Pair-wise P-value .079 .31 .11 Mean Change in PWT 1.20 0.16 0.440.31 (min) (+/−SD) (2.71) (2.62) (1.84) (2.24) Overall P value = .21

[0124] Post-Hoc Analysis of Anklebrachial Index

[0125] The initial analysis plan pre-specified that subjects having abaseline ankle brachial index (ABI)>1.2 (consistent withnon-compressible artery) be excluded from the analysis (see FIG. 9). Ina post-hoc analysis of the data, subjects having an ABI>1.2 at anytime(i.e., baseline, day 90, and/or day 180) were excluded from theanalysis. As seen in FIG. 14, this post-hoc analysis indicates that boththe single-dose and double-dose groups had a statistically significantimprovement in ABI compared to the placebo group at day 90. Thissignificance was not apparent at day 180, though the trend persisted forboth the single-dose and double-dose groups.

[0126] Efficacy Summary

[0127] Primary Efficacy Analysis

[0128] The overall P-value for the primary efficacy analysis (change inPWT at 90 days) was 0.075 (ANOVA). The overall P-value for the secondaryefficacy analysis by ANOVA of Ranks was statistically significant(P-value=0.034). The single-dose group demonstrated a 33.5% increase inPWT at day 90 versus a 20.3% increase in the double-dose group and a13.8% increase in the placebo group. Pair-wise comparison of single-doseversus placebo was statistically significant (P-value=0.026). Thistreatment effect was not maintained at day 180 using the log-transformeddata.

[0129] Secondary Efficacy Variables

[0130] Secondary efficacy variables showed no difference in PWT at day180, no difference in COT at days 90 or 180, a favorable trend in ABI inFGF-treated groups with a statistically significant change in thedouble-dose group at day 180, and no difference in calf plethysmography.Interpretation of changes in the WIQ was confounded by imbalances atbaseline. There was a trend towards improvement in the PCSS of the SF-36in the single-dose group compared to placebo at day 90; this trend wasdriven by a statistically significant difference in body-pain score.

[0131] In the pre-specified subgroups, the effect of smoking was themost interesting. The overall P-value for change in PWT at day 90 was0.007 for non-current smokers whereas it was 0.93 for current smokers.The change in PWT in the placebo group was 5.5% for non-current smokersand 29.3% for current smokers. Regression modeling suggests that smokingis a confounding variable (see post-hoc regression analysis below). Theover-representation of current smokers in the placebo group (38%, versus24% in the single-dose group and 21% in the double-dose group) made itmore difficult to detect the difference in all evaluable subjects.

[0132] Changes in PWT in non-diabetics paralleled changes seen in allevaluable subjects at days 90 and 180. Change in PWT in diabeticsparalleled changes seen in all evaluable subjects at day 90 but not atday 180. Regression modeling did not suggest that diabetes was acovariate. Changes in PWT in subjects over the median age (>68 years)were less than those in subjects under the median age at days 90 and180. Regression modeling did not suggest that age was a covariate.

[0133] The post-hoc responder analysis shows a higher percentage ofresponders in the single-dose group and a greater magnitude of effect inthe single-dose group. In addition, it suggests that the treatmenteffect persists at 180 days. The strongest predictor of response at 180days is the response at 90 days.

[0134] Post-Hoc Regression Analysis of Peak Walking Time

[0135] Regression models were used to evaluate the criteria underlyingthe use of absolute change score, relative change score, and PWT at day90 (PWT90) as analysis variables. The models are discussed below. Thispost-hoc analysis reveals that both absolute and relative change scoreshave shortcomings and do not provide the most value for assessing FGFtreatment effect in the phase II clinical trial. Using PWT90 as theanalysis variable and adjusting for baseline PWT appears to provide abetter basis, and value, for assessing the treatment effect of FGF.

[0136] Background—Assumptions in the Analysis:

[0137] I. Assume absolute change score is the correct variable to beused in the analysis. For each subject, the absolute day 90 change scorein PWT

[0138] =(PWT Day 90−PWT Baseline) or

[0139] =(PWT90−PWTB).

[0140]  Assuming that (PWT90−PWTB)=d, then PWT90=1.0*PWTB+d, (across thefull range of PWTB), and the scatter plot of PWT90 versus PWTB would be:

[0141] 1. Linear,

[0142] Slope=1.0, and

[0143] 3. Intercept would be d (unrestricted).

[0144]  FIG. 15 shows a hypothetical plot of PWT90 versus PWTB whenabsolute change score is assumed to be the correct variable.

[0145] II. Assume relative change score is the correct variable to beused in the analysis. For each subject, the relative day 90 change scorein PWT

[0146] =(PWT90/PWTB).

[0147]  Assuming that (PWT90/PWTB)=1d, then PWT90=1d*PWTB+0.0 (acrossthe full range of PWTB), and the scatter plot of PWT90 versus PWTB wouldbe:

[0148] 1. Linear,

[0149] 2. Slope would be Id (unrestricted), and

[0150] 3. Intercept would be 0.0.

[0151]  FIG. 16 shows a hypothetical plot of PWT90 versus PWTB whenrelative change score is assumed correct.

[0152] The original analysis plan used absolute change score as theanalysis variable, because there was not strong initial guidance fromPIs/consultants to use relative score, and the absolute change score wasused in the analysis of a related study directed to treatment ofcoronary artery disease (CAD). There was interest in a potential for acombined indication of FGF for CAD and PAD, which would be facilitatedby consistent use of the same analysis variable. However, the analysisplan was amended to use the Log10 relative change score(Log10(PWT90/PWTB)=(Log10 PWT90-Log10 PWTB)), as a blinded preliminaryevaluation of the skewness and kurtosis of the change score indicatedthe day 90 data did not appear to be symmetric. The day 180 PWT absolutechange score appeared (in a post-hoc analysis) to be more nearlysymmetric, i.e., the absolute change score appeared to have betterdistributional properties.

[0153] Results

[0154] The results of the post-hoc regression analysis using the modelsdescribed in Tables 15 and 16 are shown in FIGS. 17-19. FIG. 17 shows ascatter plot of PWT90 versus PWTB plus an unrestricted spline regressioncurve for each treatment group. FIG. 17 suggests that the first twocriteria for use of absolute change score (linearity and slope=1) arenot fully satisfied, and the criteria for use of relative change score(linearity and intercept=0.0) are also not satisfied. Thus, use ofabsolute change score or relative change score as the analysis variabledoes not appear to be fully consistent with the observed data.

[0155]FIG. 18 shows the same scatter plot plus curves representingregression model 2 described below. Regression models provide a moreflexible method for assessing the change in PWT at day 90 and adjustingfor baseline PWT. The curved shape of the scatter plot suggests that aregression model that produces curves will better represent or fit thestudy data. To achieve a curved shape, regression models would havePWT90 as the analysis variable, and predictor variables that includePWTB and (PWTB)². Other baseline variables such as smoking status canalso be included in the regression model, if they are importantconfounders.

[0156] Table 15 shows three regression models with PWT90 as the analysisvariable and adjusted for PWTB. All models also are adjusted for center(site). Model 1 for PWT90 includes only the predictor variable PWTB butis more flexible than using the absolute change score because model 1can have any slope (i.e., model 1 does not require the slope to be 1.0).Model 1 better fits the data than either the absolute or relative changescores.

[0157] Model 2 for PWT90 includes both PWTB and (PWTB)² as predictorvariables, and accommodates the curve shape of the scatter plot (seeFIG. 18). Model 2 appears to provide a better fit to the study data thanmodel 1, as seen by:

[0158] 1) a p-value=0.027 for the inclusion of PWTB² in the model, and

[0159] 2) an increased R²=0.52 compared to model 1, which represents astatistically significant improvement in R²=0.025).

[0160] Model 3 for PWT90 includes smoking status (at baseline), PWTB,and (PWTB)², and adjusts for current smoking status. TABLE 15 Regressionmodels for PWT90. All regression models are adjusted for Center (Site).1 2 3 Variables Trt. Single Trt. Single Trt. Single in Model Trt. DoubleTrt. Double Trt. Double PWTB PWTB PWTB (straight lines) PWTB² PWTB²(curves) Smoker (curves) Variable Single: 69.8 Single: 71.5 Single: 79.3Coefficients Double: 56.7 Double: 61.8 Double: 71.5 PWTB: 0.986 PWTB:1.917 PWTB: 1.93 PWTB²: −0.0012 PWTB²: −0.0012 Smoker: −23.8 p-value forSingle: 0.032 Single: 0.027 Single: 0.015 each variable Double: 0.096Double: 0.067 Double: 0.037 PWTB: <0.0001 PWTB: <0.0001 PWTB: <0.0001PWTB²: 0.026 PWTB²: 0.022 Smoker: 0.14 Trt. Effect (seconds) S-Placebo69.8 71.5 79.3 D-Placebo 56.7 61.8 71.5 Model R² 0.50 0.52 0.53Estimated Placebo value for PWT90 when Non- Smk PWTB equals Smk Smk 100sec. 139 sec.  75  33  57 300 sec. 336 sec. 363 323 347 600 sec. 632sec. 615 578 602

[0161] The three regression models with PWT90 suggest:

[0162] The single-dose group had a statistically significant improvementin PWT at day 90, with pairwise p-values of 0.032, 0.027, and 0.015 (formodels 1, 2, and 3, respectively).

[0163] The single-dose group had an average increase in PWT over theplacebo group of 69.8, 71.7, and 79.3 seconds.

[0164] The double-dose group had a trend or statistically significantimprovement in PWT at day 90, with pairwise p-values of 0.096, 0.0678,and 0.037.

[0165] The double-dose group had an average increase in PWT over theplacebo group of 56.7, 61.8, and 71.5 seconds; and thus the double-dosegroup is more similar to the single-dose group in increased PWT than tothe placebo group.

[0166] The regression models explain 50% or more of the variation in PWTat day 90.

[0167]FIG. 19 shows the scatter plot of PWT180 versus PWTB plus anunrestricted spline regression curve for each treatment group. The shapeof the data also do not support a slope of 1 or an intercept of 0.0. Theshape of the day 180 PWT data are only slightly curved.

[0168] Table 16 shows the three regression models for PWT180 as theanalysis variable and adjusted for PWTB, and other baseline subjectcharacteristics. All three models indicate similar results, with thesingle-dose group having a 22- to 26-second benefit over the placebogroup, and the double-dose group apparently not different than theplacebo group. TABLE 16 Regression models for PWT180. All regressionmodels are adjusted for Center (Site). 1 2 3 Variables Trt. Single Trt.Single Trt. Single in Model Trt. Double Trt. Double Trt. Double PWTBPWTB PWTB (straight lines) PWTB² PWTB² (curves) Smoker (curves) VariableSingle: 22.3 Single: 22.7 Single: 25.9 Coefficients Double: −7.4 Double:−5.8 Double: −1.7 PWTB: 1.2 PWTB: 1.4 PWTB: 1.4 PWTB²: −0.003 PWTB²:−0.0003 Smoker: −11.4 p-value for Single: 0.514 Single: 0.506 Single:0.454 each variable Double: 0.836 Double: 0.872 Double: 0.964 PWTB:<0.0001 PWTB: <0.0031 PWTB: 0.0029 PWTB²: 0.630 PWTB²: 0.608 Smoker:0.508 Trt. Effect (seconds) S-Placebo 22.3 22.7 25.9 D-Placebo −7.4 −5.8−1.7 Model R² 0.57 0.57 0.57 Estimated Placebo value for PWT90 when Non-PWTB equals Smk Smk 100 sec. 157 141 123 134 300 sec. 393 395 381 392600 sec. 745 731 723 734

[0169] In summary, the post-hoc regression analysis using the PWT at day90 as the outcome variable and adjusting for PWT at baseline showed anincrease of about 70 seconds (1.16 minutes) over placebo in thesingle-dose group, and about 57 seconds (0.95 minutes) in thedouble-dose group (p=.032, .096, respectively). Allowing therelationship to be non-linear (i.e., curved) increased the treatmenteffect to about 72 seconds (1.19 minutes) in the single-dose group, andabout 62 seconds (1.03 minutes) in the double-dose group (p=.027, .067,respectively). Adjusting for smoking status further increased thetreatment effect to about 79 seconds (1.32 minutes) in the single-dosegroup, and about 72 seconds (1.19 minutes) in the double-dose group(p=0.015, 0.035, respectively).

[0170] Conclusions from Phase II Clinical Trial

[0171] This study defined an effective dose, route, and regimen fortreatment of PAD with rFGF-2. A single-dose of 30 μg/kg rFGF-2 givenintra-arterially improved PWT of PAD patients. Administering adouble-dose of rFGF-2 was not better than administering a single-dose ofFGF-2. The magnitude of benefit in PWT was greater than 1 minute, withthe duration of benefit observed at both 3 and 6 months. In fact, about40% of the patients receiving the single-dose treatment experienced anincrease in PWT of greater than 2 minutes at both day 90 and day 180,compared with only about 22% of patients receiving placebo and about 26%of patients receiving a double dose of rFGF-2. The data indicated thatthose patients who responded at Day 90 were more likely to respond atDay 180.

[0172] Thus, repeated dosing with rFGF-2 is feasible where necessarywithout compromising patient safety. The beneficial effect on PWT seenat both day 90 and day 180 with a single dose of rFGF-2 coupled with thesafety of multiple dosing offers a method for providing prolongedtherapeutic benefit to PAD patients. This can be achieved, for example,by administering to a patient a therapeutically effective dose at day 1,and subsequent therapeutically effective doses as clinically needed,i.e., as symptoms recur.

Example 3 Phase III PAD Clinical Trial

[0173] A phase III, multicenter (up to 50 sites), double-blind,placebo-controlled, dose-optimization study is conducted. The primaryobjective of this trial is to evaluate safety and efficacy of anintra-arterial (IA) infusion of 3.0 μg/kg or 30.0 μg/kg rFGF-2 versusplacebo in peripheral artery disease (PAD) subjects with moderate tosevere claudication. The trial enrolls 450 subjects (150 per arm) withmoderate to severe claudication limiting exercise. Inclusion criteriaand exclusion criteria are shown in Table 17. Sample size may beadjusted based on DSMB evaluation of variability of peak walking time at90 days after 225 subjects are enrolled. TABLE 17 Synopsis of Phase IIIClinical Trial. Inclu- Male or female ≧40 years of age sion History ofmoderate to severe claudication limiting exercise for Cri- >6 monthsteria ABI >0.3 and <0.8 in the index limb at rest or >20% decrease afterexercise Peripheral angiogram (contrast or MRA) within 4 monthsconfirming >70% obstruction of one or more infra- inguinal vessels,patent femoral inflow bilaterally, and absence of hemodynamicallysignificant supra-inguinal obstruction Able to exercise >1 minute but<12 minutes on two Gardner treadmill exercise tests. Exercise time mustbe limited by claudication at baseline. Duplicate tests will beperformed >24 hours but not >2 weeks apart. The difference betweenbaseline exercise times must be ≦20% of their mean. Medically stable for3 months with laboratory parameters within clinically acceptable rangefor required procedures. Serum creatinine ≦2.2 mg/dL and urineprotein/creatinine ratio <.3 Willing and able to give written informedconsent Exclu- Peripheral Artery Disease sion History of rest pain,non-healing ulcer, or gangrene Cri- within 3 months teria Evidence ofhemodynamically significant aorto-iliac obstructive disease Peripheralrevascularization (PTI or surgery) within 3 months Malignancy History ofmalignancy within past 5 years (exceptions: curatively treated basalcell carcinoma, squamous cell carcinoma of the skin in sun-exposedareas, or carcinoma of the cervix) Evidence or suspicion of malignancyafter screening according to ACS guidelines Ocular conditionsProliferative retinopathy or moderate or severe non-proliferativeretinopathy Maculopathy with choroidal neovascularization or macularedema Intra-ocular surgery within 3 months Cardiovascular conditionsMyocardial infarction, CABG, PTCA within 3 months Transient ischemicattack or stroke within 3 months General medical conditions: Pregnancy,nursing mothers Participation in clinical trials of otherinvestigational agents, intra-arterial devices, or procedures, for whichfollow-up visits have not been completed History of organtransplantation Any combined condition which makes the subjectunsuitable for participation in the opinion of the Investigator, e.g.,concurrent medical illness which limits life expectancy to <12 months,psychosis, severe mental retardation, inability to communicate withstudy personnel, drug or alcohol abuse Diagnosis of primary pulmonaryhypertension, restrictive or obstructive cardiomyopathy, activevasculitis Previous participation in any therapeutic angiogenesis trialwith any investigational agent unless subject received placebo

[0174] The study drug, rFGF-2 having the sequence shown in FIG. 2 (SEQID NO:2), is contained at 0.35 or 3.5 mg/mL in a lyophilized powder, tobe reconstituted with normal saline; it is formulated in 10 mM sodiumcitrate, 1 mM EDTA, 10 mM dithiothreitol (DTT), 4% glycine, 1% glucoseat pH 6.0. Treatment consists of infusion of 20 mL at 1 mL/min ofplacebo, 3.0μg/kg or 30.0 μg/kg rFGF-2, divided equally between twolegs, via the common femoral artery. Assignment to treatments israndomized 1:1:1 placebo: 3.0 μg/kg rFGF-2: 30.0 μg/kg rFGF-2. Bloodwill be drawn at baseline and at the end of the infusion for analysis ofplasma concentration of FGF-2. Each subject is observed in the hospitalfor 6 hours following study drug administration and followed as anoutpatient at specified intervals for 180 days.

[0175] Patients are monitored for acute safety variables includingsystolic hypotension associated with IA infusion and any evidence ofallergic reactions, as well as frequency and severity of adverse events,changes in laboratory parameters (especially urine protein), evidence ofretinal toxicity, and evidence of seroconversion (antibody formation).DSMB will review SAEs and abnormal laboratory tests during enrollment.

[0176] Primary efficacy variable is change from baseline in peak walkingtime (PWT) at 90 days as measured by Gardner graded exercise test time,adjusted for baseline PWT, smoking status, and center. Secondaryefficacy is established based on the following parameters:

[0177] Change from baseline in PWT at 45, 135, and 180 days adjusted forbaseline PWT, smoking status, and center;

[0178] Change from baseline in claudication onset time (COT) at 45, 90,135, and 180 days;

[0179] Change from baseline in anklebrachial index pressure (ABI) at 45,90, 135, and 180 days;

[0180] Change from baseline in severity of claudication, distance,speed, and stair climbing scores of the WIQ at 45, 90, 135, and 180days;

[0181] Change from baseline in physical component summary score (PCSS)of the SF-36 at 45, 90, 135, and 180 days; and

[0182] Percentage of responders at 90 and 180 days.

[0183] Potential substudies include plethysmography, muscle biopsy, andMR spectroscopy. General protocol and information tests at follow-upvisits are shown in Table 18. TABLE 18 General Schedule of Events.Primary Screen Dosing Endpoint Termination DAY DAY DAY DAY DAY DAY DAYTests −45 to −1 1 15 45 90 135 180 Complete History and X Physical Examconsistent with ACS guidelines Limited History and X X X X X PhysicalExam Telephone FU X 12-lead ECG X X X X Chest x-ray X PSA (males only);X serum pregnancy (females only); screen for HIV, hepatitis and drugs ofabuse if appropriate Laboratory Tests (CBC, X X X X X X X platelets,chemistry¹, urine²) FGF-2 Antibodies X X X X X X Gardner graded exerciseX, X X X X X test for PWT and COT; ABIs Ophthalmologic exam; X X fundusphotography only if evidence of retinopathy Quality of Life: WIQ, X X XX X SF-36 Limited Angiogram X Study Drug X Administration Blood Samplefor FGF-2 X and Infusion Solution Sample Concomitant X X X X X X XMedications Adverse events (AEs) X X X X X X

[0184] Statistical Analysis

[0185] Data are analyzed with an intent to treat analysis using ANOVA ofRanks with last value after baseline carried forward for missing data,or lowest rank for subjects without a post-baseline PWT assessment,adjusting for baseline PWT, smoking status, and center.

Example 4 Influence of FGF-2 Dosing Regimens on Collateral Blood Flow inRats With Peripheral Arterial Insufficiency

[0186] A study was undertaken to compare the efficacy of three routes ofFGF-2 administration (intra-arterial [positive control], intramuscular,and a route used in humans) to increase collateral blood in rats withexperimental peripheral arterial insufficiency.

[0187] Previous animal model studies have demonstrated that bFGF iseffective at improving collateral blood flow to the distal calf musclesfollowing bilateral femoral artery occlusion (Yang et al. (1996) Circ.Res. 79:62-69; Yang and Feng (2000) Am. J. Physiol. 278:H85-H93). Theimproved blood flow from ˜50 ml/min/100 g to 70-80 ml/min/100 g waspossible due to a significant decrease in vascular resistance of thecollateral vessels of the upper thigh. The increase in collateral bloodflow to the calf muscles correlates well with an angiographic scoreobtained from x-ray images of the thigh arterial tree. Upper thighcollateral vessels are the major site of resistance in the circuitfollowing occlusion of the femoral arteries (Yang et al. (1996) Circ.Res. 79:62-69). It is not likely that de novo synthesis of newcapillaries (angiogenesis) could develop into large conduit vessels andaccount for this vascular response. Rather, the extent of the resistancechange and the short time for vascular development to occur (16 days)makes it probable that enlargement of existing vessels was the primarychange contributing to the greater blood flow. This increase incollateral blood flow with bFGF is also found in aged rats (Yang andFeng (2000) Am. J. Physiol. 278:H85-H93) and enhanced with physicalactivity (Yang et al. (1998) Am. J. Physiol. 274:H2053-H2061).

[0188] A variety of routes and regimens of bFGF administration have beenshown effective at increasing collateral blood flow in animal models.These include close-arterial systemic, and subcutaneous routes withbolus, short-term and relatively long-term delivery regimens achieved byinjections or timed infusions with osmotic pumps (Yang and Feng (2000)Am. J. Physiol. 278:H85-H93). While these protocols have been useful todemonstrate the efficacy of bFGF, some of these regimens are notappropriate for patient management in therapeutic angiogenesis. Further,the value of repeated administration of bFGF at timed intervals inpatients is expected, but not feasible with many of the procedures usedin previous experimental studies. For example, it would be extremelyvaluable to establish the efficacy of intramuscular injections of bFGF.However, it is presently unclear whether direct intramuscular injectionsof bFGF impart targeted or generalized improvement in collateral bloodflow. Thus, the purpose of the present pilot study was to evaluate theefficacy of FGF-2 administration via intramuscular injections and aclinically relevant protocol used in therapeutic angiogenesis.

[0189] Experimental Design: Animals with peripheral arterialinsufficiency were divided into four groups: Group 1 Intra-arterial, 14day continuous infusion, FGF-2 N = 6 (5 μg/kg/day) Group 2 Vehicle groupcomprised of: group 2a 14 day intra-arterial continuous N = 2 infusionof vehicle group 2b Single intra-arterial injection of vehicle N = 2group 2c Intramuscular, single bolus of vehicle N = 4 Group 3 SingleIntra-arterial Injection group 3a Dose 1 (1.5 μg/kg total) N = 6 group3b Dose 2 (15 μg/kg total) N = 6 group 3c Dose 3 (30 μg/kg total) N = 6Group 4 Intramuscular Injection group 4a Dose 1 (0.15 μg/kg total) N = 6group 4b Dose 2 (1.5 μg/kg total) N = 6 group 4c Dose 3 (15 μg/kg total)N = 6

[0190] Because of distinct delivery routes, the study was not performedin a completely blinded manner. However, animals within a delivery routereceived the treatments of dose (i.e., vehicle, infusion, dose 1, dose2, or dose 3) in a randomly-assigned blinded manner.

[0191] General Protocol:

[0192] Adult Sprague-Dawley rats (approximately 325 g) were conditionedto the treadmill by walking for 5-10 min twice daily for 5 days. Toinitiate the experiment, the animals were subjected to bilateral femoralartery occlusion (cf. Methods). On the same day, animals began atwo-week treatment according to the treatment groups described above. Onday 16 of the experiment, collateral-dependent blood was determinedwhile the rats were running on a motor-driven treadmill. Followingcompletion of the data set, the results were pooled according totreatment group, and the results were analyzed statistically by ANOVA.It was expected that vehicle-treated animals from each of the deliveryroutes could be grouped into one reference control group. However, datawere assessed to determine whether the intramuscular injection treatmenthad introduced a systematic response, for example, caused by aninflammatory response in the muscle.

[0193] Peripheral Arterial Insufficiency Model:

[0194] Bilateral ligation of the femoral artery is designed to establishperipheral arterial insufficiency without impairing resting muscle bloodflow. The high blood flow reserve of muscle is markedly reduced whileresidual muscle blood flow is sufficient to support resting blood flowneeds; e.g., compare (Yang et al. (1990) J. Appl. Physiol 69:1353-1359;Yang and Terjung(1993) J. Appl, Physiol. 75(1):452-457; Mackie andTerjung (1983) Am. J. Physiol. 245:H265-H275). Thus, there is no ‘restpain’ nor complications leading to pathological changes, tissuenecrosis, or gangrene observed with more proximal vascular obstructions(Chleboun and Martin (1994) Aust. N. Z. J. Surg. 64:202-207). It isrecognized that these surgically treated animals do not represent thebroad spectrum of peripheral arterial insufficiency found clinically.Rather, this model is characteristic of large vessel occlusive diseasethat often presents itself with symptoms of intermittent claudication.

[0195] Methods:

[0196] Animal Care.

[0197] Adult Sprague Dawley rats (approximately 325 g), obtained fromTaconic Farms, Germantown, N.Y., were housed in a temperature controlledroom (20+1° C.), with a 12hr/12hr light/dark cycle. Animals were givenPurina Rat Chow and tap water ad libitum. Previous studies haveestablished that a complete data set of approximately 12 animals pergroup is necessary to definitively evaluate treatment effects (Yang etal. (1990) J. Appl. Physiol 69:1353-1359). Thus, the present work was apilot study to assess the general response for future consideration ofthe half of the study. Since ·90% of rats received from the supplier arewilling runners (to be randomly assigned to treatment groups) and someattrition occurs in the conduct of the experiment, it was expected thatn=5-6 per experimental group would be obtained.

[0198] FGF-2 Delivery.

[0199] FGF-2 delivery was initiated/achieved at the time of femoralartery ligation by: a) a 14-day continuous intra-arterial infusion; b) asingle intramuscular injection; or c) a single intra-arterial injectionas follows.

[0200] For the positive control, a group of 8 rats received a 14-dayinfusion from an in-dwelling pump/catheter (rate=0.5 μL/hr). Thecatheter was placed such that the infusion was delivered upstream of theligation point in the femoral artery of one hindlimb. Six of the ratsreceived FGF-2 at a dose of 5 μg/kg/day for 14 days for a total of 70μg/kg; the other two received vehicle alone (PBS). To allow for deadspaces, filling the pump, filling the tubing, etc., a final volume of0.435 mL of pump solution for each rat was calculated based on theinitial rat weight of approximately 325 g. A separate pump solutionaliquot was prepared for each rat. Just prior to use, 43.5 μL of sodiumcitrate and 7 μL of glycerol was added to each aliquot, so that thevolume of FGF-2 solution or PBS in each of the prepared tubes was0.435-0.0435-0.007=0.385 mL. For the FGF aliquots, the concentration ofFGF-2 was thus 152.5 μg/mL.

[0201] A second group of rats received vehicle or FGF-2 with a singleintra-arterial injection. This injection was given over the span of 10minutes, into the femoral artery of one hindlimb, upstream of the pointof ligation. The volume to be injected was 0.35 mL. Six rats received1.5 μg/kg FGF-2 total dose; six received 15 μg/kg FGF-2; six received 30μg/kg FGF-2; and two received vehicle alone.

[0202] A third group of rats received a single intramuscular injection.This injection was split between two sites in the medial hamstring onone hindlimb, in the region of collateral formation. The volume to beinjected was 100 μL per site, for a total volume of 0.2 mL. Six ratsreceived 0. 15 μg/kg FGF-2 total dose; six received 1.5 μg/kg FGF-2; sixreceived 15 μg/kg FGF-2; and four received vehicle alone.

[0203] Ligation Surgery.

[0204] Under ether anesthesia each femoral artery was isolated justdistal to the inguinal ligament. A ligature was placed tightly aroundthe femoral vessel to assure total obstruction to blood flow. Topicalantibiotic powder (Neo-Predef, Upjohn) was placed on the wound prior toclosure with skin clips. The surgical procedure was brief, was achievedwith a 100% success rate, and the animals recovered rapidly. As doneroutinely (Yang and Terjung(1993) J. Appl, Physiol. 75(1):452-457; Yanget al. (1995a) Circ. Res. 76.448-456; Yang et al. (1995b) Am J. Physiol.268:H1 174-HI 180), visual inspection at the time of autopsy verifiedthe success of surgery.

[0205] Blood Flow Determination during Treadmill Running In Vivo.

[0206] Muscle blood flow was determined in a blinded manner, utilizingradiolabeled microspheres during treadmill running, as used extensively(Yang and Terjung(1993) J. Appl, Physiol. 75(1):452-457; Mackie andTerjung (1983) Am. J. Physiol. 245:H265-H275; Mathien and Terjung (1986)Am. J. Physiol. 245:H1050-H1059; Mathien and Terjung (1990) Am J.Physiol. 258:H759-H765; Yang et al. (1990) J. Appl. Physiol69:1353-1359; Yang et al. (1995) Circ. Res. 76.448-456; Yang et al.(1995) Am J. Physiol. 268:H1174-H1180; Yang et al. (1996) Circ. Res.79:62-69). Microspheres (15μm diameter), labeled with ⁸⁵Sr or ¹⁴¹Ce (−10mCi/g), were obtained commercially (NEN, Boston) in a suspension of 10%dextran containing 0.05% Tween 80. A well-mixed suspension ofmicrospheres was carefully infused into the arch of the aorta, followedby a saline flush, over a 15-20 second period. Direct comparisons ofinjection sites (left ventricle versus aortic arch) gave the same bloodflows to the kidneys and hindlimb muscles. Approximately 360,000 sphereswere infused to establish an adequate microsphere distribution and topermit statistical assurance in the data (Mackie and Terjung (1983) Am.J. Physiol. 245:H265-H275; Mathien and Terjung (1986) Am. J. Physiol.245:H1050-H1059; Mathien and Terjung (1990) Am J. Physiol.258:H759-H765). Typically, there were well in excess of 400 microspheresper individual muscle sample during exercise. Withdrawal of thereference blood sample at 500 μl/min (Sage Instruments Model 355 pump)from the caudal artery was initiated 10 sec prior to infusion ofmicrospheres and continued for approximately 100 sec. It has been foundthat reference blood samples, withdrawn from the right carotid artery,femoral artery, and caudal artery, match within 5-10% of each other(Mackie and Terjung (1983) Am. J. Physiol. 245:H265-H275; Unpub. Obs).

[0207] Two blood flow determinations were performed in each animal, at amoderate and higher treadmill speed, to establish peak vascularconductance of the muscle so that the upstream collateral resistance inthe upper thigh becomes rate-determining for downstream blood flow tothe calf muscles. This is achieved, since muscle contraction (exercise)is the most powerful stimulus for vasodilation. Following exercise, therats were sacrificed by an overdose of pentobarbital and the tissuesamples obtained as described below, and counted (LKB Universal GammaCounter), with the reference blood sample, to a 1% counting error.Bilateral sections of kidney (middle 3rd) were taken to verify theadequacy of microsphere mixing. Appropriate corrections were made forbackground and isotopic spillover. Blood flows (ml/min/100 g) werecalculated as follows:

FLOW=(CPM _(T) ×FLOW _(RBS)×100)/(CPM _(RBS) ×Wt _(T))

[0208] where T is tissue and RBS is the reference blood sample.

[0209] Heart rate and arterial pressure were continuously monitoredduring exercise.

[0210] Surgical Procedures for Blood Flow Determination. Surgicalpreparation for in vivo blood flow determination was a modification ofthat described by Laughlin et al. (1982) J. Appl. Physiol 52:1629-1635.Animals were anesthetized with ketamine/ACE-promazine (100 mg/0.5 mg perkg) and a catheter was inserted into the right carotid artery to thearch of the aorta for later infusion of the microspheres. A catheter (PE50 tapered) was also placed into the caudal artery for the withdrawal ofblood sample. Both catheters were filled with saline containing heparin(100 IU/ml), led under the skin, and exteriorized at the back of theneck. Each incision site was sutured and covered with 1% xylocaineointment (Astra Pharm.). Consistent with the experience of others(Gleeson and Baldwin (1981)J. Appl. Physiol. 50:1205-1211), after 3-4 hrfollowing placement of the catheters the rats were alert, willing torun, and exhibited normal exercise tolerance.

[0211] Muscle Sections.

[0212] All tissues of the hindlimb from the hip socket distal, aredissected, weighed, and counted for radioactivity. Muscles (Greene(1963) Anatomy of the Rat, New York Hafner Pub. Co.) include: bicepsfemoris, semitendinosus, semimembranosus, caudofemoralis, adductorgroup, gluteus group, tensor fascias latae, quadriceps group, soleus,plantaris, gastrocnemius, tibialis anterior, extensor digitorurm longus,deep lateral and posterior crural muscles. The tibia, fibula, femur andfoot are also weighed and counted. In addition, sections composedprimarily of fast-twitch red fibers (deep lateral quadriceps and deeplateral gastrocnemius), fast-twitch white fibers (superficial quadricepsand superficial medial gastrocnemius), and slow-twitch red fibers(soleus) are obtained. A thorough biochemical, physiological andmorphological characterization of these muscle fiber sections is known(cf. Saltin and Gollaick (1983) In: Handbook of Physiology—SkeletalMuscle, Am. Physiol. Soc., pp. 555-631). Blood flow to the diaphragmwill also be determined to follow the response of an active muscle thatis not subject to ligation.

[0213] Statistical Procedures.

[0214] Statistical evaluation employing repeated measures analysis ofvariance, Tukey's comparison of means, and a t-test were performed asappropriate (Steel and Torrie (1960) Principles & Procedures ofStatistics, McGraw-Hill, New York).

[0215] Results:

[0216] Intramuscular administration of rFGF-2 increased blood flow in adose-dependent manner (FIG. 20). When administered intra-arterially, the15μg/kg rFGF-2 dose was just as efficacious as the 30 μg/kg dose.Continuous infusion over a 14-day period did not provide a significantlydifferent efficacy relative to the single IA infusion or single IMinjection mode of administration.

[0217] These data demonstrate efficacy of single IM injection and singleIA infusion of rFGF-2 in enhancing blood flow to the hindlimb quartersin a PAD animal model.

[0218] All publications and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference. Although theforegoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, itwill be obvious that certain changes and modifications may be practicedwithin the scope of the appended embodiment.

1 9 1 441 DNA Bos taurus CDS (1)...(441) 1 cca gcc cta cca gaa gat gggggg tcc ggg gcc ttc cca cca ggg cac 48 Pro Ala Leu Pro Glu Asp Gly GlySer Gly Ala Phe Pro Pro Gly His 1 5 10 15 ttc aaa gat cca aaa cga ctatat tgt aaa aac ggg ggg ttc ttc cta 96 Phe Lys Asp Pro Lys Arg Leu TyrCys Lys Asn Gly Gly Phe Phe Leu 20 25 30 cga atc cac cca gat ggg cga gtagat ggg gta cga gaa aaa tcc gat 144 Arg Ile His Pro Asp Gly Arg Val AspGly Val Arg Glu Lys Ser Asp 35 40 45 cca cac atc aaa cta caa cta caa gccgaa gaa cga ggg gta gta tcc 192 Pro His Ile Lys Leu Gln Leu Gln Ala GluGlu Arg Gly Val Val Ser 50 55 60 atc aaa ggg gta tgt gcc aac cga tat ctagcc atg aaa gaa gat ggg 240 Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu AlaMet Lys Glu Asp Gly 65 70 75 80 cga cta cta gcc tcc aaa tgt gta acc gatgaa tgt ttc ttc ttc gaa 288 Arg Leu Leu Ala Ser Lys Cys Val Thr Asp GluCys Phe Phe Phe Glu 85 90 95 cga cta gaa tcc aac aac tat aac acc tat cgatcc cga aaa tat tcc 336 Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg SerArg Lys Tyr Ser 100 105 110 tcc tgg tat gta gcc cta aaa cga acc ggg caatat aaa cta ggg cca 384 Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln TyrLys Leu Gly Pro 115 120 125 aaa acc ggg cca ggg caa aaa gcc atc cta ttccta cca atg tcc gcc 432 Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe LeuPro Met Ser Ala 130 135 140 aaa tcc taa 441 Lys Ser * 145 2 146 PRT Bostaurus 2 Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His1 5 10 15 Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe PheLeu 20 25 30 Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys SerAsp 35 40 45 Pro His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val ValSer 50 55 60 Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu AspGly 65 70 75 80 Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe PhePhe Glu 85 90 95 Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg LysTyr Ser 100 105 110 Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr LysLeu Gly Pro 115 120 125 Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe LeuPro Met Ser Ala 130 135 140 Lys Ser 145 3 441 DNA Homo sapiens CDS(1)...(441) 3 ccc gcc ttg ccc gag gat ggc ggc agc ggc gcc ttc ccg cccggc cac 48 Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro GlyHis 1 5 10 15 ttc aag gac ccc aag cgg ctg tac tgc aaa aac ggg ggc ttcttc ctg 96 Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe PheLeu 20 25 30 cgc atc cac ccc gac ggc cga gtt gac ggg gtc cgg gag aag agcgac 144 Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp35 40 45 cct cac atc aag cta caa ctt caa gca gaa gag aga gga gtt gtg tct192 Pro His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser 5055 60 atc aaa gga gtg tgt gct aac cgt tac ctg gct atg aag gaa gat gga240 Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly 6570 75 80 aga tta ctg gct tct aaa tgt gtt acg gat gag tgt ttc ttt ttt gaa288 Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu 8590 95 cga ttg gaa tct aat aac tac aat act tac cgg tca agg aaa tac acc336 Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Thr 100105 110 agt tgg tat gtg gca ctg aaa cga act ggg cag tat aaa ctt gga tcc384 Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser 115120 125 aaa aca gga cct ggg cag aaa gct ata ctt ttt ctt cca atg tct gct432 Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala 130135 140 aag agc tga 441 Lys Ser * 145 4 146 PRT Homo sapiens 4 Pro AlaLeu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His 1 5 10 15 PheLys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu 20 25 30 ArgIle His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp 35 40 45 ProHis Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser 50 55 60 IleLys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly 65 70 75 80Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu 85 90 95Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Thr 100 105110 Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser 115120 125 Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala130 135 140 Lys Ser 145 5 468 DNA Bos taurus CDS (1)...(468) 5 atg gcagcc ggg agc atc acc acg ctg cca gcc cta cca gaa gat ggg 48 Met Ala AlaGly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15 ggg tccggg gcc ttc cca cca ggg cac ttc aaa gat cca aaa cga cta 96 Gly Ser GlyAla Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30 tat tgt aaaaac ggg ggg ttc ttc cta cga atc cac cca gat ggg cga 144 Tyr Cys Lys AsnGly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg 35 40 45 gta gat ggg gtacga gaa aaa tcc gat cca cac atc aaa cta caa cta 192 Val Asp Gly Val ArgGlu Lys Ser Asp Pro His Ile Lys Leu Gln Leu 50 55 60 caa gcc gaa gaa cgaggg gta gta tcc atc aaa ggg gta tgt gcc aac 240 Gln Ala Glu Glu Arg GlyVal Val Ser Ile Lys Gly Val Cys Ala Asn 65 70 75 80 cga tat cta gcc atgaaa gaa gat ggg cga cta cta gcc tcc aaa tgt 288 Arg Tyr Leu Ala Met LysGlu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95 gta acc gat gaa tgt ttcttc ttc gaa cga cta gaa tcc aac aac tat 336 Val Thr Asp Glu Cys Phe PhePhe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110 aac acc tat cga tcc cgaaaa tat tcc tcc tgg tat gta gcc cta aaa 384 Asn Thr Tyr Arg Ser Arg LysTyr Ser Ser Trp Tyr Val Ala Leu Lys 115 120 125 cga acc ggg caa tat aaacta ggg cca aaa acc ggg cca ggg caa aaa 432 Arg Thr Gly Gln Tyr Lys LeuGly Pro Lys Thr Gly Pro Gly Gln Lys 130 135 140 gcc atc cta ttc cta ccaatg tcc gcc aaa tcc taa 468 Ala Ile Leu Phe Leu Pro Met Ser Ala LysSer * 145 150 155 6 155 PRT Bos taurus 6 Met Ala Ala Gly Ser Ile Thr ThrLeu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15 Gly Ser Gly Ala Phe Pro ProGly His Phe Lys Asp Pro Lys Arg Leu 20 25 30 Tyr Cys Lys Asn Gly Gly PhePhe Leu Arg Ile His Pro Asp Gly Arg 35 40 45 Val Asp Gly Val Arg Glu LysSer Asp Pro His Ile Lys Leu Gln Leu 50 55 60 Gln Ala Glu Glu Arg Gly ValVal Ser Ile Lys Gly Val Cys Ala Asn 65 70 75 80 Arg Tyr Leu Ala Met LysGlu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95 Val Thr Asp Glu Cys PhePhe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110 Asn Thr Tyr Arg SerArg Lys Tyr Ser Ser Trp Tyr Val Ala Leu Lys 115 120 125 Arg Thr Gly GlnTyr Lys Leu Gly Pro Lys Thr Gly Pro Gly Gln Lys 130 135 140 Ala Ile LeuPhe Leu Pro Met Ser Ala Lys Ser 145 150 155 7 474 DNA Homo sapiens CDS(1)...(468) 7 atg gca gcc ggg agc atc acc acg ctg ccc gcc ttg ccc gaggat ggc 48 Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu AspGly 1 5 10 15 ggc agc ggc gcc ttc ccg ccc ggc cac ttc aag gac ccc aagcgg ctg 96 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys ArgLeu 20 25 30 tac tgc aaa aac ggg ggc ttc ttc ctg cgc atc cac ccc gac ggccga 144 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg35 40 45 gtt gac ggg gtc cgg gag aag agc gac cct cac atc aag cta caa ctt192 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu 5055 60 caa gca gaa gag aga gga gtt gtg tct atc aaa gga gtg tgt gct aac240 Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn 6570 75 80 cgt tac ctg gct atg aag gaa gat gga aga tta ctg gct tct aaa tgt288 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 8590 95 gtt acg gat gag tgt ttc ttt ttt gaa cga ttg gaa tct aat aac tac336 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100105 110 aat act tac cgg tca agg aaa tac acc agt tgg tat gtg gca ctg aaa384 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115120 125 cga act ggg cag tat aaa ctt gga tcc aaa aca gga cct ggg cag aaa432 Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys 130135 140 gct ata ctt ttt ctt cca atg tct gct aag agc tga ttttaa 474 AlaIle Leu Phe Leu Pro Met Ser Ala Lys Ser * 145 150 155 8 155 PRT Homosapiens 8 Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu AspGly 1 5 10 15 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro LysArg Leu 20 25 30 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro AspGly Arg 35 40 45 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys LeuGln Leu 50 55 60 Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val CysAla Asn 65 70 75 80 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu AlaSer Lys Cys 85 90 95 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu SerAsn Asn Tyr 100 105 110 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp TyrVal Ala Leu Lys 115 120 125 Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys ThrGly Pro Gly Gln Lys 130 135 140 Ala Ile Leu Phe Leu Pro Met Ser Ala LysSer 145 150 155 9 9 PRT Bos taurus 9 Met Ala Ala Gly Ser Ile Thr Thr Leu1 5

That which is claimed:
 1. A method for treating peripheral arterydisease in a patient, said method comprising administering to saidpatient a therapeutically effective amount of fibroblast growth factor(FGF), wherein said therapeutically effective amount of FGF is dividedinto two doses and a single dose is administered into each leg of saidpatient within a one hour period.
 2. The method of claim 1, wherein saidFGF is administered by intra-arterial infusion (IA) into at least oneartery of each leg of said patient.
 3. The method of claim 2, whereinsaid FGF is administered into the common femoral artery of each leg ofsaid patient.
 4. The method of claim 3, wherein said FGF is administeredvia bilateral delivery using a catheter.
 5. The method of claim 3,wherein said FGF is administered via direct IA infusion into the commonfemoral artery of each leg of said patient.
 6. The method of claim 1,wherein said FGF is administered by one or more intramuscular (IM)injections.
 7. The method according to claim 1, wherein said peripheralartery disease is evidenced by claudication.
 8. The method according toclaim 7, wherein said patient has critical limb ischemia.
 9. The methodof claim 1, wherein said FGF is FGF-2.
 10. The method of claim 9,wherein said FGF-2 is a recombinant molecule.
 11. The method of claim10, wherein said FGF-2 comprises the sequence set forth in FIG. 2 (SEQID NO:2), FIG. 3 (SEQ ID NO:4), FIG. 4 (SEQ ID NO:6), FIG. 5 (SEQ IDNO:8) or an angiogenically active fragment or mutein thereof.
 12. Themethod of claim 11, wherein said mutein comprises an FGF-2 moleculewherein at least one constituent cysteine residue is replaced by aneutral amino acid.
 13. The method of claim 12, wherein the neutralamino acid is serine or threonine.
 14. The method of claim 11, whereinsaid FGF-2 is administered simultaneously with another molecule selectedfrom the group consisting of heparin and other proteoglycan.
 15. Themethod of claim 14, wherein said heparin is a low molecular weightmolecule.
 16. The method of claim 14, wherein said heparin isunfractionated heparin.
 17. The method of claim 11, wherein said FGF-2is administered within about 5 minutes to about 60 minutes of heparin orproteoglycan administration to said patient.
 18. The method of claim 17,wherein said FGF-2 is administered within about 20 minutes to about 30minutes of heparin or other proteoglycan administration to said patient.19. The method of claim 11, wherein said FGF-2 is administered in theabsence of administering a molecule selected from the group consistingof heparin and other proteoglycan.
 20. The method of claim 11, whereinsaid therapeutically effective amount of FGF-2 is administered to saidpatient once in a 24 hour period.
 21. The method of claim 11, whereinsaid therapeutically effective amount of FGF-2 is administered to saidpatient once a week.
 22. The method of claim 11, wherein saidtherapeutically effective amount of FGF-2 is administered to saidpatient once a month, once every 2 months, once every 3 months, onceevery four months, once every five months, or once every six months. 23.The method of claim 11, wherein said therapeutically effective amount ofFGF-2 is administered as an adjunct to vascular surgery, mechanicalbypass surgery, angioplasty, or angiogram.
 24. The method of claim 11,wherein said therapeutically effective amount of said FGF-2 or saidangiogenically active fragment or mutein thereof is about
 0. 1 μg/kg toabout 1 μg/kg.
 25. The method of claim 11, wherein said therapeuticallyeffective amount of said FGF-2 or said angiogenically active fragment ormutein thereof is about 1 μg/kg to about 3 μg/kg.
 26. The method ofclaim 11, wherein said therapeutically effective amount of said FGF-2 orsaid angiogenically active fragment or mutein thereof is about 3 μg/kgto about 5 μg/kg.
 27. The method of claim 11, wherein saidtherapeutically effective amount of said FGF-2 or said angiogenicallyactive fragment or mutein thereof is about 5 μg/kg to about 7 μg/kg. 28.The method of claim 11, wherein said therapeutically effective amount ofsaid FGF-2 or said angiogenically active fragment or mutein thereof isabout 7 μg/kg to about 9 μg/kg.
 29. The method of claim 11, wherein saidtherapeutically effective amount of said FGF-2 or said angiogenicallyactive fragment or mutein thereof is about 9 μg/kg to about 10 μg/kg.30. The method of claim 11, wherein said therapeutically effectiveamount of said FGF-2 or said angiogenically active fragment or muteinthereof is about 10 μg/kg to about 15 μg/kg.
 31. The method of claim 11,wherein said therapeutically effective amount of said FGF-2 or saidangiogenically active fragment or mutein thereof is about 15 μg/kg toabout 20 μg/kg.
 32. The method of claim 11, wherein said therapeuticallyeffective amount of said FGF-2 or said angiogenically active fragment ormutein thereof is about 20 μg/kg to about 25 μg/kg.
 33. The method ofclaim 11, wherein said therapeutically effective amount of said FGF-2 orsaid angiogenically active fragment or mutein thereof is about 25 μg/kgto about 30 μg/kg.
 34. The method of claim 11, wherein saidtherapeutically effective amount of said FGF-2 or said angiogenicallyactive fragment or mutein thereof is about 30 μg/kg to about 40 μg/kg.35. The method of claim 11, wherein said therapeutically effectiveamount of said FGF-2 or said angiogenically active fragment or muteinthereof is about 40 μg/kg to about 50 μg/kg.
 36. The method of claim 11,wherein said therapeutically effective amount of said FGF-2 or saidangiogenically active fragment or mutein thereof is about 4 μg to about0.3 mg.
 37. The method of claim 11, wherein said therapeuticallyeffective amount of said FGF-2 or said angiogenically active fragment ormutein thereof is about 0.3 mg to about 3.5 mg.
 38. The method of claim37, wherein said therapeutically effective amount of said FGF-2 or saidangiogenically active fragment or mutein thereof is about 1.0 to about2.0mg.
 39. The method of claim 37, wherein said therapeuticallyeffective amount of said FGF-2 or said angiogenically active fragment ormutein thereof is about 2.0 to about 3.5mg.
 40. The method of claim 9,wherein said FGF-2 is administered to said patient by intra-arterial(IA) or intravenous (IV) infusion.
 41. The method of claim 9, whereinsaid FGF-2 is administered to said patient by one or more intramuscular(IM) injections.
 42. The method of claim 9, wherein said FGF-2 isadministered to said patient by subcutaneous (SC) injection.
 43. Themethod of claim 9, wherein said administering of FGF-2 provides animprovement in peak walking time (PWT) in said patient relative to PWTin the absence of said administering of FGF-2.
 44. The method of claim9, wherein said administering of FGF-2 provides an improvement inanklebrachial index (ABI) in said patient relative to ABI in the absenceof said administering of FGF-2.
 45. The method of claim 9, wherein saidadministering of FGF-2 results in a reduction in body pain.
 46. Themethod of claim 9, wherein said administering of FGF-2 improves stairclimbing ability.
 47. The method of claim 9, wherein said administeringof FGF-2 reduces the severity of claudication.
 48. A method for treatingperipheral artery disease in a patient, said method comprisingadministering to said patient a therapeutically effective amount offibroblast growth factor-2 (FGF-2), wherein said therapeuticallyeffective amount is about 0.1 μg/kg to about 9.9 μμg/kg.
 49. The methodof claim 48, wherein said therapeutically effective amount of FGF-2 isadministered as part of a pharmaceutical composition.
 50. The method ofclaim 49, wherein said pharmaceutical composition is a stabilizedFGF-2-DTT formulation.
 51. The method of claim 48, wherein said FGF-2 isadministered simultaneously with another molecule selected from thegroup consisting of heparin and other proteoglycan.
 52. The method ofclaim 48, wherein said therapeutically effective amount of said FGF-2 orsaid angiogenically active fragment or mutein thereof is about 0.1 μg/kgto about 1 μg/kg.
 53. The method of claim 48, wherein saidtherapeutically effective amount of said FGF-2 or said angiogenicallyactive fragment or mutein thereof is about 1 μg/kg to about 3 μg/kg. 54.The method of claim 48, wherein said therapeutically effective amount ofsaid FGF-2 or said angiogenically active fragment or mutein thereof isabout 3 μg/kg to about 5 μg/kg.
 55. The method of claim 48, wherein saidtherapeutically effective amount of said FGF-2 or said angiogenicallyactive fragment or mutein thereof is about 5 μg/kg to about 7 μg/kg. 56.The method of claim 48, wherein said therapeutically effective amount ofsaid FGF-2 or said angiogenically active fragment or mutein thereof isabout 7 μg/kg to about 8 μg/kg.
 57. The method of claim 48, wherein saidtherapeutically effective amount of said FGF-2 or said angiogenicallyactive fragment or mutein thereof is about 8 μg/kg to about 9 μg/kg. 58.The method of claim 48, wherein said therapeutically effective amount ofsaid FGF-2 or said angiogenically active fragment or mutein thereof isabout 9 μg/kg to about 9.9 μg/kg.
 59. The method of claim 48, whereinsaid therapeutically effective amount of said FGF-2 or saidangiogenically active fragment or mutein thereof is about 7.0 μg toabout 0.7 mg.
 60. The method of claim 59, wherein said therapeuticallyeffective amount of said FGF-2 or said angiogenically active fragment ormutein thereof is about 9.0 μg to about 0.5 mg.
 61. The method of claim60, wherein said therapeutically effective amount of said FGF-2 or saidangiogenically active fragment or mutein thereof is about 0.1 mg toabout 0.4 mg.
 62. The method of claim 61, wherein said therapeuticallyeffective amount of said FGF-2 or said angiogenically active fragment ormutein thereof is about 0.1 mg to about 0.2 mg.
 63. The method of claim48, wherein said FGF-2 is administered to said patient by intra-arterial(IA) or intravenous (IV) infusion.
 64. The method of claim 48, whereinsaid FGF-2 is administered to said patient by one or more intramuscular(IM) injections.
 65. A method for improving peak walking time in apatient with intermittent claudication, said method comprisingadministering to said patient a therapeutically effective amount offibroblast growth factor (FGF), wherein said therapeutically effectiveamount of FGF is divided into two doses and a single dose isadministered into each leg of said patient within a one hour period. 66.The method of claim 65, wherein said FGF is FGF-2.
 67. The method ofclaim 66, wherein said therapeutically effective amount of said FGF-2 isabout 0.1 μg/kg to about 1 μg/kg.
 68. The method of claim 66, whereinsaid therapeutically effective amount of said FGF-2 is about 1 μg/kg toabout 3 μg/kg.
 69. The method of claim 66, wherein said therapeuticallyeffective amount of said FGF-2 is about 3 μg/kg to about 5 μg/kg. 70.The method of claim 66, wherein said therapeutically effective amount ofsaid FGF-2 is about 5 μg/kg to about 9 μg/kg.
 71. The method of claim66, wherein said therapeutically effective amount of said FGF-2 is about9 μg/kg to about 10 μg/kg.
 72. The method of claim 66, wherein saidtherapeutically effective amount of said FGF-2 is about 10 μg/kg toabout 20 μg/kg.
 73. The method of claim 66, wherein said therapeuticallyeffective amount of said FGF-2 is about 20 μg/kg to about 30 μg/kg. 74.A method for improving ankle-brachial index in a patient withintermittent claudication, said method comprising administering to saidpatient a therapeutically effective amount of fibroblast growth factor(FGF), wherein said therapeutically effective amount of FGF is dividedinto two doses and a single dose is administered into each leg of saidpatient within a one hour period.
 75. The method of claim 74, whereinsaid FGF is FGF-2.
 76. The method of claim 75, wherein saidtherapeutically effective amount of said FGF-2 is about 0.1 μg/kg toabout 1 μg/kg.
 77. The method of claim 75, wherein said therapeuticallyeffective amount of said FGF-2 is about 1 μg/kg to about 3 μg/kg. 78.The method of claim 75, wherein said therapeutically effective amount ofsaid FGF-2 is about 3 μg/kg to about 5 μg/kg.
 79. The method of claim75, wherein said therapeutically effective amount of said FGF-2 is about5 μg/kg to about 9 μg/kg.
 80. The method of claim 75, wherein saidtherapeutically effective amount of said FGF-2 is about 9 μg/kg to about10 μg/kg.
 81. The method of claim 75, wherein said therapeuticallyeffective amount of said FGF-2 is about 10 μg/kg to about 20 μg/kg. 82.The method of claim 75, wherein said therapeutically effective amount ofsaid FGF-2 is about 20 μg/kg to about 30 μg/kg.